Primer composition, kit and method for detecting mutation of gene related to benign and malignant thyroid nodules

Abstract

The present application relates to a primer composition, kit and method for detecting benign and malignant thyroid nodule related gene mutations, wherein the primer composition comprises primer pairs for detecting DNA mutations and RNA fusion variations of benign and malignant thyroid nodule related genes. The primer pairs can be amplified individually, and more preferably, the DNA and cDNA templates to be tested can be multiplexed with high efficiency, high sensitivity and high specificity in the same PCR reaction system and reaction program. Subsequently, by constructing a library and sequencing, the gene mutation of the relevant region can be obtained, thereby assisting the clinical diagnosis of benign and malignant thyroid nodules. Furthermore, by connecting specific nucleotide sequences to the 5' end of the upstream primer and the 3' end of the corresponding downstream primer in the primer composition in a proper proportion, the uniformity and specificity of the multiplex PCR amplification can be further improved, and the nucleic acid input for detection and the minimum detection limit of mutation frequency (as low as 0.5%) can be effectively reduced.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology and relates to gene mutation detection, specifically to primer compositions, kits and methods for detecting gene mutations related to benign and malignant thyroid nodules. Background Technology

[0002] Currently, ultrasound-guided fine-needle aspiration cytology (US-FNAB) is considered the gold standard for preoperative diagnosis of thyroid tumors in clinical practice. However, 10%-40% of samples cannot be definitively diagnosed as benign or malignant using US-FNAB. For thyroid nodules whose benign or malignant nature cannot be determined, molecular testing for hotspot genes in thyroid tumors should be added to the US-FNAB examination.

[0003] Conventional tumor molecular detection generally employs hybridization capture library preparation, with two most common methods: The first is NGS large-panel detection, which can cover hundreds of genes / locus sites in a single test. However, the large detection area leads to high sequencing costs and expensive testing, and its lack of focus on genes related to thyroid malignancies results in data waste. The second method is customized small-panel detection for thyroid tumors, which can detect 10-15 genes related to thyroid tumors (including fixed hotspot fusion gene sites). The number of genes detected is relatively small, and the initial nucleic acid input required for hybridization capture library preparation is high (at least 50-100 ng). Most thyroid fine-needle aspiration samples are small in volume and have low nucleic acid concentrations, making it difficult to meet the detection requirements. Furthermore, hybridization capture library preparation is complex and has a long experimental process, with the entire detection cycle taking 7-8 days. Small panels are prone to off-target effects during capture and elution, resulting in limited detection sensitivity and thus lacking clinical universality.

[0004] Besides hybridization capture library preparation, amplicon library preparation is currently used for molecular detection of tumors. Compared with hybridization capture library preparation, this method requires a lower initial amount of nucleic acid. However, amplicon library preparation has disadvantages in terms of PCR reaction uniformity. Conventional multiplex amplicon reactions have amplification bias, tending to preferentially amplify short fragments, which leads to non-specific amplification. The amplification of a large number of non-specific fragments in the same system will consume PCR enzymes and substrates, resulting in reduced amplification efficiency of the target fragment and poor overall amplification uniformity. Currently, there is no technical route for molecular detection of thyroid fine needle biopsy samples using the above-mentioned library preparation methods.

[0005] Therefore, there is a need in the market for a molecular detection method that has good uniformity, high sensitivity, low requirements for nucleic acid input, and price advantage, to help differentiate between benign and malignant thyroid nodules in clinical practice, reduce the surgical resection rate of thyroid nodules, and reduce the economic burden on patients while facilitating precise diagnosis and treatment. Summary of the Invention

[0006] Based on this, the purpose of the present invention is to provide primer compositions, kits and methods for detecting gene mutations related to benign and malignant thyroid nodules. The primer compositions have good amplification uniformity, high sensitivity and high specificity, and can effectively reduce the amount of nucleic acid input for detection and the minimum detection limit of gene mutation frequency.

[0007] A first aspect of the present invention is to provide a primer composition for detecting gene mutations related to benign and malignant thyroid nodules, comprising primer pairs for detecting DNA mutations in the BRAF and KRAS genes;

[0008] The primer pair for detecting BRAF gene DNA mutations is selected from at least one of the following: an upstream primer with the sequence shown in SEQ ID NO.5 and a downstream primer with the sequence shown in SEQ ID NO.6; an upstream primer with the sequence shown in SEQ ID NO.7 and a downstream primer with the sequence shown in SEQ ID NO.8; an upstream primer with the sequence shown in SEQ ID NO.9 and a downstream primer with the sequence shown in SEQ ID NO.10;

[0009] The primer pair for detecting KRAS gene DNA mutations is selected from at least one of the following: an upstream primer with the sequence shown in SEQ ID NO. 121 and a downstream primer with the sequence shown in SEQ ID NO. 122; an upstream primer with the sequence shown in SEQ ID NO. 123 and a downstream primer with the sequence shown in SEQ ID NO. 124; an upstream primer with the sequence shown in SEQ ID NO. 125 and a downstream primer with the sequence shown in SEQ ID NO. 126; an upstream primer with the sequence shown in SEQ ID NO. 127 and a downstream primer with the sequence shown in SEQ ID NO. 128.

[0010] In some embodiments, the primer composition further includes at least one primer pair for detecting DNA mutations in the NRAS, TERT, TP53 and RET genes;

[0011] The primer pair for detecting NRAS gene DNA mutations is selected from at least one of the following: the upstream primer with the sequence shown in SEQ ID NO. 129 and the downstream primer with the sequence shown in SEQ ID NO. 130; the upstream primer with the sequence shown in SEQ ID NO. 131 and the downstream primer with the sequence shown in SEQ ID NO. 132; the upstream primer with the sequence shown in SEQ ID NO. 133 and the downstream primer with the sequence shown in SEQ ID NO. 134; the upstream primer with the sequence shown in SEQ ID NO. 135 and the downstream primer with the sequence shown in SEQ ID NO. 136;

[0012] The primer pair for detecting TERT gene DNA mutations includes: an upstream primer with the sequence shown in SEQ ID NO. 385 and a downstream primer with the sequence shown in SEQ ID NO. 386;

[0013] The primer pairs for detecting TP53 gene DNA mutations are selected from at least one of the following: the upstream primer with the sequence shown in SEQ ID NO. 387 and the downstream primer with the sequence shown in SEQ ID NO. 388; the upstream primer with the sequence shown in SEQ ID NO. 389 and the downstream primer with the sequence shown in SEQ ID NO. 390; the upstream primer with the sequence shown in SEQ ID NO. 391 and the downstream primer with the sequence shown in SEQ ID NO. 392; the upstream primer with the sequence shown in SEQ ID NO. 393 and the downstream primer with the sequence shown in SEQ ID NO. 394; the upstream primer with the sequence shown in SEQ ID NO. 395 and the downstream primer with the sequence shown in SEQ ID NO. 396; the upstream primer with the sequence shown in SEQ ID NO. 397 and the downstream primer with the sequence shown in SEQ ID NO. 398; the upstream primer with the sequence shown in SEQ ID NO. 399 and the downstream primer with the sequence shown in SEQ ID NO. 400; the upstream primer with the sequence shown in SEQ ID NO. 401 and the downstream primer with the sequence shown in SEQ ID NO. 402; the primer with the sequence shown in SEQ ID NO. 39 ...0; the primer with the sequence shown in SEQ ID NO. 401 and the downstream primer with the sequence shown in SEQ ID NO. 402; the primer with the sequence shown in SEQ ID NO. 399 and the downstream primer with the sequence shown in SEQ ID NO. 399; the primer with the sequence shown in SEQ ID NO. 399 and the downstream primer with the sequence shown in The upstream primer shown in NO.403 and the downstream primer shown in SEQ ID NO.404;

[0014] The primer pair for detecting RET gene DNA mutations is selected from at least one of the following: the upstream primer with the sequence shown in SEQ ID NO. 357 and the downstream primer with the sequence shown in SEQ ID NO. 358; the upstream primer with the sequence shown in SEQ ID NO. 359 and the downstream primer with the sequence shown in SEQ ID NO. 360; the upstream primer with the sequence shown in SEQ ID NO. 361 and the downstream primer with the sequence shown in SEQ ID NO. 362; the upstream primer with the sequence shown in SEQ ID NO. 363 and the downstream primer with the sequence shown in SEQ ID NO. 364; the upstream primer with the sequence shown in SEQ ID NO. 365 and the downstream primer with the sequence shown in SEQ ID NO. 366; the upstream primer with the sequence shown in SEQ ID NO. 367 and the downstream primer with the sequence shown in SEQ ID NO. 368; the upstream primer with the sequence shown in SEQ ID NO. 369 and the downstream primer with the sequence shown in SEQ ID NO. 370; the upstream primer with the sequence shown in SEQ ID NO. 371 and the downstream primer with the sequence shown in SEQ ID NO. 372; the ... primer with the sequence shown in SEQ ID NO. 371 and the downstream primer with the sequence shown in SEQ ID NO. 372; the primer with the sequence shown in SEQ ID NO. 369 and the downstream primer with the sequence shown in SEQ ID NO. 370; the primer with the sequence shown in SEQ ID NO. 371 and the downstream primer with the sequence shown in The upstream primer shown in SEQ ID NO. 373 and the downstream primer with the sequence shown in SEQ ID NO. 374; the upstream primer with the sequence shown in SEQ ID NO. 375 and the downstream primer with the sequence shown in SEQ ID NO. 376; the upstream primer with the sequence shown in SEQ ID NO. 377 and the downstream primer with the sequence shown in SEQ ID NO. 378; the upstream primer with the sequence shown in SEQ ID NO. 379 and the downstream primer with the sequence shown in SEQ ID NO. 380; the upstream primer with the sequence shown in SEQ ID NO. 381 and the downstream primer with the sequence shown in SEQ ID NO. 382; the upstream primer with the sequence shown in SEQ ID NO. 383 and the downstream primer with the sequence shown in SEQ ID NO. 384.

[0015] In some embodiments, the primer composition further includes at least one primer pair for detecting DNA mutations in the CDKN2A, DICER1, EIF1AX, GNAS, HRAS, PIK3CA, PTEN, TSHR, ARAF, AKT1, CTNNB1, and RAF1 genes;

[0016] The primer pairs for detecting CDKN2A gene DNA mutations are selected from at least one of the following: the upstream primer and the downstream primer shown in SEQ ID NO. 11; the upstream primer and the downstream primer shown in SEQ ID NO. 14; the upstream primer and the downstream primer shown in SEQ ID NO. 15; the upstream primer and the downstream primer shown in SEQ ID NO. 16; the upstream primer and the downstream primer shown in SEQ ID NO. 17; the upstream primer and the downstream primer shown in SEQ ID NO. 18; the upstream primer and the downstream primer shown in SEQ ID NO. 19; the upstream primer and the downstream primer shown in SEQ ID NO. 20; the upstream primer and the downstream primer shown in SEQ ID NO. 21; the upstream primer and the downstream primer shown in SEQ ID NO. 22; the upstream primer and the downstream primer shown in SEQ ID NO. 23; the upstream primer and the downstream primer shown in SEQ ID NO. 24; the upstream primer and the downstream primer shown in SEQ ID NO. 25; the upstream primer and the downstream primer shown in SEQ ID NO. 26; the upstream primer and the downstream primer shown in SEQ ID NO. 27; the upstream primer and the downstream primer shown in SEQ ID NO. 18; the upstream primer and the downstream primer shown in SEQ ID NO. 20; the upstream primer and the downstream primer shown in SEQ ID NO. 21; the upstream primer and the downstream primer shown in SEQ ID NO. 22; the upstream primer and the downstream primer shown in SEQ ID NO. 23; the upstream primer and the downstream primer shown in SEQ ID NO. 24; the upstream primer and the downstream primer shown in SEQ ID NO. 25; the upstream primer and the downstream primer shown in SEQ ID NO. 26; the upstream primer and the downstream primer shown in SEQ ID NO. 27; the upstream primer and the downstream primer shown in SEQ ID NO. 20; the upstream primer and the downstream primer shown in SEQ ID NO. 20; the upstream primer and the downstream primer shown in SEQ ID NO. 20; the The downstream primer shown in NO. 28; the upstream primer with the sequence shown in SEQ ID NO. 29 and the downstream primer with the sequence shown in SEQ ID NO. 30; the upstream primer with the sequence shown in SEQ ID NO. 31 and the downstream primer with the sequence shown in SEQ ID NO. 32;

[0017] The primer pair for detecting DICER1 gene DNA mutations is selected from at least one of the following: an upstream primer with the sequence shown in SEQ ID NO.41 and a downstream primer with the sequence shown in SEQ ID NO.42; an upstream primer with the sequence shown in SEQ ID NO.43 and a downstream primer with the sequence shown in SEQ ID NO.44; an upstream primer with the sequence shown in SEQ ID NO.45 and a downstream primer with the sequence shown in SEQ ID NO.46;

[0018] The primer pairs for detecting EIF1AX gene DNA mutations are selected from at least one of the following: the upstream primer and the downstream primer shown in SEQ ID NO. 47; the upstream primer and the downstream primer shown in SEQ ID NO. 48; the upstream primer and the downstream primer shown in SEQ ID NO. 49; the upstream primer and the downstream primer shown in SEQ ID NO. 50; the upstream primer and the downstream primer shown in SEQ ID NO. 51; the upstream primer and the downstream primer shown in SEQ ID NO. 52; the upstream primer and the downstream primer shown in SEQ ID NO. 53; the upstream primer and the downstream primer shown in SEQ ID NO. 54; the upstream primer and the downstream primer shown in SEQ ID NO. 55; the upstream primer and the downstream primer shown in SEQ ID NO. 56; the upstream primer and the downstream primer shown in SEQ ID NO. 57; the upstream primer and the downstream primer shown in SEQ ID NO. 58; the upstream primer and the downstream primer shown in SEQ ID NO. 59; the upstream primer and the downstream primer shown in SEQ ID NO. 60; the upstream primer and the downstream primer shown in SEQ ID NO. 61; the upstream primer and the downstream primer shown in SEQ ID NO. 62; the upstream primer and the downstream primer shown in SEQ ID NO. 63; the upstream primer and the downstream primer shown in SEQ ID NO. 64; the upstream primer and the downstream primer shown in SEQ ID NO. 65; the upstream primer and the downstream primer shown in SEQ ID NO. 66; the upstream primer and the downstream primer shown in SEQ ID NO. 67; the upstream primer and the downstream primer shown in SEQ ID NO. 68; the upstream primer and the downstream primer shown in SEQ ID NO. 69; the upstream primer and the downstream primer shown in SEQ ID NO. 60; the upstream primer and the downstream primer shown in SEQ ID NO. 61; the upstream primer and the downstream primer shown in SEQ ID NO. 62; the upstream primer and the downstream primer shown in SEQ ID NO. 63; the upstream The following primers are listed: the downstream primer shown in SEQ ID NO. 64; the upstream primer shown in SEQ ID NO. 65 and the downstream primer shown in SEQ ID NO. 66; the upstream primer shown in SEQ ID NO. 67 and the downstream primer shown in SEQ ID NO. 68; the upstream primer shown in SEQ ID NO. 69 and the downstream primer shown in SEQ ID NO. 70; the upstream primer shown in SEQ ID NO. 71 and the downstream primer shown in SEQ ID NO. 72; the upstream primer shown in SEQ ID NO. 73 and the downstream primer shown in SEQ ID NO. 74; the upstream primer shown in SEQ ID NO. 75 and the downstream primer shown in SEQ ID NO. 76; the upstream primer shown in SEQ ID NO. 77 and the downstream primer shown in SEQ ID NO. 78; the upstream primer shown in SEQ ID NO. 79 and the downstream primer shown in SEQ ID NO. 80; the upstream primer shown in SEQ ID NO. 81 and the downstream primer shown in SEQ ID NO. 82; the upstream primer shown in SEQ ID NO. 64 and the downstream primer shown in SEQ ID NO. 70; the upstream primer shown in SEQ ID NO. 71 and the downstream primer shown in SEQ ID NO. 72; the upstream primer shown in SEQ ID NO. 73 and the downstream primer shown in SEQ ID NO. 74; the upstream primer shown in SEQ ID NO. 75 and the downstream primer shown in SEQ ID NO. 76; the upstream primer shown in SEQ ID NO. 77 and the downstream primer shown in SEQ ID NO. 78; the upstream primer shown in SEQ ID NO. 79 and the downstream primer shown in SEQ ID NO. 80; the upstream primer shown in SEQ ID NO. 81 and the downstream primer shown in SEQ ID NO. 82; the upstream primer shown in SEQ ID NO. 74 and the downstream primer shown in SEQ ID NO. 80; the upstream primer shown in SEQ ID NO. 81 and The upstream primer shown in SEQ ID NO. 83 and the downstream primer with the sequence shown in SEQ ID NO. 84; the upstream primer with the sequence shown in SEQ ID NO. 85 and the downstream primer with the sequence shown in SEQ ID NO. 86; the upstream primer with the sequence shown in SEQ ID NO. 87 and the downstream primer with the sequence shown in SEQ ID NO.The downstream primer shown in SEQ ID NO. 88; the upstream primer and the downstream primer shown in SEQ ID NO. 90; the upstream primer and the downstream primer shown in SEQ ID NO. 91; the upstream primer and the downstream primer shown in SEQ ID NO. 92; the upstream primer and the downstream primer shown in SEQ ID NO. 93; the upstream primer and the downstream primer shown in SEQ ID NO. 94; the upstream primer and the downstream primer shown in SEQ ID NO. 95; the upstream primer and the downstream primer shown in SEQ ID NO. 96; the upstream primer and the downstream primer shown in SEQ ID NO. 97; the upstream primer and the downstream primer shown in SEQ ID NO. 98; the upstream primer and the downstream primer shown in SEQ ID NO. 99; the upstream primer and the downstream primer shown in SEQ ID NO. 100; the upstream primer and the downstream primer shown in SEQ ID NO. 101; the upstream primer and the downstream primer shown in SEQ ID NO. 102; the upstream primer and the downstream primer shown in SEQ ID NO. 103; the upstream primer and the downstream primer shown in SEQ ID NO. 104; the upstream primer and the downstream primer shown in SEQ ID NO. 105; the downstream primer and the downstream primer shown in SEQ ID NO. 98; the upstream primer and the downstream primer shown in SEQ ID NO. 100; the upstream primer and the downstream primer shown in SEQ ID NO. 102; the upstream primer and the downstream primer shown in SEQ ID NO. 103; the upstream primer and the downstream primer shown in SEQ ID NO. 104; the upstream primer and the downstream primer shown in SEQ ID NO. 105; the upstream primer and the downstream primer shown in SEQ ID NO. 100; the upstream primer and the downstream primer shown in SEQ ID NO. 100; the upstream primer and the downstream primer shown in SEQ ID NO. 102; the upstream primer and the downstream primer shown in SEQ ID NO. 103; the upstream primer and the downstream primer shown in SEQ ID NO. The downstream primer shown in SEQ ID NO. 106; the upstream primer with the sequence shown in SEQ ID NO. 107; and the downstream primer with the sequence shown in SEQ ID NO. 108.

[0019] The primer pair for detecting GNAS gene DNA mutations is selected from at least one of the following: an upstream primer with a sequence as shown in SEQ ID NO. 109 and a downstream primer with a sequence as shown in SEQ ID NO. 110; an upstream primer with a sequence as shown in SEQ ID NO. 111 and a downstream primer with a sequence as shown in SEQ ID NO. 112;

[0020] The primer pair for detecting HRAS gene DNA mutations is selected from at least one of the following: the upstream primer with the sequence shown in SEQ ID NO. 113 and the downstream primer with the sequence shown in SEQ ID NO. 114; the upstream primer with the sequence shown in SEQ ID NO. 115 and the downstream primer with the sequence shown in SEQ ID NO. 116; the upstream primer with the sequence shown in SEQ ID NO. 117 and the downstream primer with the sequence shown in SEQ ID NO. 118; the upstream primer with the sequence shown in SEQ ID NO. 119 and the downstream primer with the sequence shown in SEQ ID NO. 120;

[0021] The primer pairs for detecting PIK3CA gene DNA mutations are selected from at least one of the following: the upstream primer with the sequence shown in SEQ ID NO. 137 and the downstream primer with the sequence shown in SEQ ID NO. 138; the upstream primer with the sequence shown in SEQ ID NO. 139 and the downstream primer with the sequence shown in SEQ ID NO. 140; the upstream primer with the sequence shown in SEQ ID NO. 141 and the downstream primer with the sequence shown in SEQ ID NO. 142; the upstream primer with the sequence shown in SEQ ID NO. 143 and the downstream primer with the sequence shown in SEQ ID NO. 144; the upstream primer with the sequence shown in SEQ ID NO. 145 and the downstream primer with the sequence shown in SEQ ID NO. 146; the upstream primer with the sequence shown in SEQ ID NO. 147 and the downstream primer with the sequence shown in SEQ ID NO. 148; the upstream primer with the sequence shown in SEQ ID NO. 149 and the downstream primer with the sequence shown in SEQ ID NO. 150; the upstream primer with the sequence shown in SEQ ID NO. 151 and the downstream primer with the sequence shown in SEQ ID NO. 152; the ... primer with the sequence shown in SEQ ID NO. 151 and the downstream primer with the sequence shown in SEQ ID NO. 152; the primer with the sequence shown in SEQ ID NO. 149 and the downstream primer with the sequence shown in SEQ ID NO. 150; the primer with the sequence shown in SEQ ID NO. 151 and the downstream primer with the sequence The upstream primer shown in SEQ ID NO. 153 and the downstream primer with the sequence shown in SEQ ID NO. 154; the upstream primer with the sequence shown in SEQ ID NO. 155 and the downstream primer with the sequence shown in SEQ ID NO. 156; the upstream primer with the sequence shown in SEQ ID NO. 157 and the downstream primer with the sequence shown in SEQ ID NO. 158; the upstream primer with the sequence shown in SEQ ID NO. 159 and the downstream primer with the sequence shown in SEQ ID NO. 160; the upstream primer with the sequence shown in SEQ ID NO. 161 and the downstream primer with the sequence shown in SEQ ID NO. 162; the upstream primer with the sequence shown in SEQ ID NO. 163 and the downstream primer with the sequence shown in SEQ ID NO. 164; the upstream primer with the sequence shown in SEQ ID NO. 165 and the downstream primer with the sequence shown in SEQ ID NO. 166; the upstream primer with the sequence shown in SEQ ID NO. 167 and the downstream primer with the sequence shown in SEQ ID NO. 168; the upstream primer with the sequence shown in SEQ ID NO. 169 and the downstream primer with the sequence shown in SEQ ID NO. 154; the upstream primer with the sequence shown in SEQ ID NO. 155 and the downstream primer with the sequence shown in SEQ ID NO. 156; the upstream primer with the sequence shown in SEQ ID NO. 157 and the downstream primer with the sequence shown in SEQ ID NO. 158; the upstream primer with the sequence shown in SEQ ID NO. 159 and the downstream primer with the sequence shown in SEQ ID NO. 160; the upstream primer with the sequence shown in SEQ ID NO. 161 and the downstream primer with the sequence shown in SEQ ID NO. 162; the upstream primer with the sequence shown in SEQ ID NO. 163 and the downstream primer with the sequence shown in SEQ ID NO. 164; the upstream primer with the sequence shown in SEQ The downstream primer shown in SEQ ID NO. 170; the upstream primer shown in SEQ ID NO. 171 and the downstream primer shown in SEQ ID NO. 172; the upstream primer shown in SEQ ID NO. 173 and the downstream primer shown in SEQ ID NO. 174; the upstream primer shown in SEQ ID NO. 175 and the downstream primer shown in SEQ ID NO. 176; and the primer shown in SEQ ID NO.The upstream primer shown in SEQ ID NO. 177 and the downstream primer with the sequence shown in SEQ ID NO. 178; the upstream primer with the sequence shown in SEQ ID NO. 179 and the downstream primer with the sequence shown in SEQ ID NO. 180; the upstream primer with the sequence shown in SEQ ID NO. 181 and the downstream primer with the sequence shown in SEQ ID NO. 182; the upstream primer with the sequence shown in SEQ ID NO. 183 and the downstream primer with the sequence shown in SEQ ID NO. 184; the upstream primer with the sequence shown in SEQ ID NO. 185 and the downstream primer with the sequence shown in SEQ ID NO. 186; the upstream primer with the sequence shown in SEQ ID NO. 187 and the downstream primer with the sequence shown in SEQ ID NO. 188; the upstream primer with the sequence shown in SEQ ID NO. 189 and the downstream primer with the sequence shown in SEQ ID NO. 190; the upstream primer with the sequence shown in SEQ ID NO. 191 and the downstream primer with the sequence shown in SEQ ID NO. 192; the upstream primer with the sequence shown in SEQ ID NO. 19 ... The following primers are listed: the downstream primer shown in SEQ ID NO. 194; the upstream primer shown in SEQ ID NO. 195 and the downstream primer shown in SEQ ID NO. 196; the upstream primer shown in SEQ ID NO. 197 and the downstream primer shown in SEQ ID NO. 198; the upstream primer shown in SEQ ID NO. 199 and the downstream primer shown in SEQ ID NO. 200; the upstream primer shown in SEQ ID NO. 201 and the downstream primer shown in SEQ ID NO. 202; the upstream primer shown in SEQ ID NO. 203 and the downstream primer shown in SEQ ID NO. 204; the upstream primer shown in SEQ ID NO. 205 and the downstream primer shown in SEQ ID NO. 206; the upstream primer shown in SEQ ID NO. 207 and the downstream primer shown in SEQ ID NO. 208; the upstream primer shown in SEQ ID NO. 209 and the downstream primer shown in SEQ ID NO. 210; the downstream primer shown in SEQ ID NO. 194; the upstream primer shown in SEQ ID NO. 195 and the downstream primer shown in SEQ ID NO. 196; the upstream primer shown in SEQ ID NO. 197 and the downstream primer shown in SEQ ID NO. 198; the upstream primer shown in SEQ ID NO. 199 and the downstream primer shown in SEQ ID NO. 210; the upstream primer shown in SEQ ID NO. 194; the upstream primer shown in SEQ ID NO. 195 and the downstream primer shown in SEQ ID NO. 200; the upstream primer shown in SEQ ID NO. 201 and the downstream primer shown in SEQ ID NO. 202; the upstream primer shown in SEQ ID NO. 203 and the downstream primer shown in SEQ ID NO. 204; the upstream primer shown in SEQ ID NO. 205 and the downstream primer shown in SEQ ID NO. 206; the upstream primer shown in SEQ ID NO The upstream primer shown in NO. 211 and the downstream primer with the sequence shown in SEQ ID NO. 212; the upstream primer with the sequence shown in SEQ ID NO. 213 and the downstream primer with the sequence shown in SEQ ID NO. 214; the upstream primer with the sequence shown in SEQ ID NO. 215 and the downstream primer with the sequence shown in SEQ ID NO. 216; the upstream primer with the sequence shown in SEQ ID NO. 217 and the downstream primer with the sequence shown in SEQ ID NO.The downstream primer shown in SEQ ID NO. 218; the upstream primer shown in SEQ ID NO. 219 and the downstream primer shown in SEQ ID NO. 220; the upstream primer shown in SEQ ID NO. 221 and the downstream primer shown in SEQ ID NO. 222; the upstream primer shown in SEQ ID NO. 223 and the downstream primer shown in SEQ ID NO. 224; the upstream primer shown in SEQ ID NO. 225 and the downstream primer shown in SEQ ID NO. 226; the upstream primer shown in SEQ ID NO. 227 and the downstream primer shown in SEQ ID NO. 228; the upstream primer shown in SEQ ID NO. 229 and the downstream primer shown in SEQ ID NO. 230; the upstream primer shown in SEQ ID NO. 231 and the downstream primer shown in SEQ ID NO. 232; the upstream primer shown in SEQ ID NO. 233 and the downstream primer shown in SEQ ID NO. 234; the upstream primer shown in SEQ ID NO. 218 and the downstream primer shown in SEQ ID NO. 220; the upstream primer shown in SEQ ID NO. 221 and the downstream primer shown in SEQ ID NO. 222; the upstream primer shown in SEQ ID NO. 223 and the downstream primer shown in SEQ ID NO. 234; the upstream primer shown in SEQ ID NO. 229 and the downstream primer shown in SEQ ID NO. 230; the upstream primer shown in SEQ ID NO. 231 and the downstream primer shown in SEQ ID NO. 232; the upstream primer shown in SEQ ID NO. 23 ... The upstream primer shown in NO. 235 and the downstream primer with the sequence shown in SEQ ID NO. 236;

[0022] The primer pairs for detecting PTEN gene DNA mutations are selected from at least one of the following: the upstream primer with the sequence shown in SEQ ID NO. 237 and the downstream primer with the sequence shown in SEQ ID NO. 238; the upstream primer with the sequence shown in SEQ ID NO. 239 and the downstream primer with the sequence shown in SEQ ID NO. 240; the upstream primer with the sequence shown in SEQ ID NO. 241 and the downstream primer with the sequence shown in SEQ ID NO. 242; the upstream primer with the sequence shown in SEQ ID NO. 243 and the downstream primer with the sequence shown in SEQ ID NO. 244; the upstream primer with the sequence shown in SEQ ID NO. 245 and the downstream primer with the sequence shown in SEQ ID NO. 246; the upstream primer with the sequence shown in SEQ ID NO. 247 and the downstream primer with the sequence shown in SEQ ID NO. 248; the upstream primer with the sequence shown in SEQ ID NO. 249 and the downstream primer with the sequence shown in SEQ ID NO. 250; the upstream primer with the sequence shown in SEQ ID NO. 251 and the downstream primer with the sequence shown in SEQ ID NO. 252; the ... primer with the sequence shown in SEQ ID NO. 251 and the downstream primer with the sequence shown in SEQ ID NO. 252; the primer with the sequence shown in SEQ ID NO. 249 and the downstream primer with the sequence shown in SEQ ID NO. 250; the primer with the sequence shown in SEQ ID NO. 251 and the downstream primer with the sequence shown in The upstream primer shown in SEQ ID NO. 253 and the downstream primer with the sequence shown in SEQ ID NO. 254; the upstream primer with the sequence shown in SEQ ID NO. 255 and the downstream primer with the sequence shown in SEQ ID NO. 256; the upstream primer with the sequence shown in SEQ ID NO. 257 and the downstream primer with the sequence shown in SEQ ID NO. 258; the upstream primer with the sequence shown in SEQ ID NO. 259 and the downstream primer with the sequence shown in SEQ ID NO. 260; the upstream primer with the sequence shown in SEQ ID NO. 261 and the downstream primer with the sequence shown in SEQ ID NO. 262; the upstream primer with the sequence shown in SEQ ID NO. 263 and the downstream primer with the sequence shown in SEQ ID NO. 264; the upstream primer with the sequence shown in SEQ ID NO. 265 and the downstream primer with the sequence shown in SEQ ID NO. 266; the upstream primer with the sequence shown in SEQ ID NO. 267 and the downstream primer with the sequence shown in SEQ ID NO. 268; the upstream primer with the sequence shown in SEQ ID NO. 269 and the downstream primer with the sequence shown in SEQ ID NO. 254; the upstream primer with the sequence shown in SEQ ID NO. 255 and the downstream primer with the sequence shown in SEQ ID NO. 256; the upstream primer with the sequence shown in SEQ ID NO. 257 and the downstream primer with the sequence shown in SEQ ID NO. 258; the upstream primer with the sequence shown in SEQ ID NO. 259 and the downstream primer with the sequence shown in SEQ ID NO. 260; the upstream primer with the sequence shown in SEQ ID NO. 261 and the downstream primer with the sequence shown in SEQ ID NO. 262; the upstream primer with the sequence shown in SEQ ID NO. 263 and the downstream primer with the sequence shown in SEQ ID NO. 264; the upstream primer with the sequence shown in SEQ The downstream primer shown in SEQ ID NO. 270; the upstream primer shown in SEQ ID NO. 271 and the downstream primer shown in SEQ ID NO. 272; the upstream primer shown in SEQ ID NO. 273 and the downstream primer shown in SEQ ID NO. 274; the upstream primer shown in SEQ ID NO. 275 and the downstream primer shown in SEQ ID NO. 276; and the primer shown in SEQ ID NO.The upstream primer shown in SEQ ID NO. 277 and the downstream primer with the sequence shown in SEQ ID NO. 278; the upstream primer with the sequence shown in SEQ ID NO. 279 and the downstream primer with the sequence shown in SEQ ID NO. 280; the upstream primer with the sequence shown in SEQ ID NO. 281 and the downstream primer with the sequence shown in SEQ ID NO. 282; the upstream primer with the sequence shown in SEQ ID NO. 283 and the downstream primer with the sequence shown in SEQ ID NO. 284; the upstream primer with the sequence shown in SEQ ID NO. 285 and the downstream primer with the sequence shown in SEQ ID NO. 286; the upstream primer with the sequence shown in SEQ ID NO. 287 and the downstream primer with the sequence shown in SEQ ID NO. 288; the upstream primer with the sequence shown in SEQ ID NO. 289 and the downstream primer with the sequence shown in SEQ ID NO. 290; the upstream primer with the sequence shown in SEQ ID NO. 291 and the downstream primer with the sequence shown in SEQ ID NO. 292; the upstream primer with the sequence shown in SEQ ID NO. 29 ... The downstream primer shown in SEQ ID NO. 294; the upstream primer shown in SEQ ID NO. 295 and the downstream primer shown in SEQ ID NO. 296; the upstream primer shown in SEQ ID NO. 297 and the downstream primer shown in SEQ ID NO. 298; the upstream primer shown in SEQ ID NO. 299 and the downstream primer shown in SEQ ID NO. 300; the upstream primer shown in SEQ ID NO. 301 and the downstream primer shown in SEQ ID NO. 302; the upstream primer shown in SEQ ID NO. 303 and the downstream primer shown in SEQ ID NO. 304; the upstream primer shown in SEQ ID NO. 305 and the downstream primer shown in SEQ ID NO. 306; the upstream primer shown in SEQ ID NO. 307 and the downstream primer shown in SEQ ID NO. 308; the upstream primer shown in SEQ ID NO. 309 and the downstream primer shown in SEQ ID NO. 310; the downstream primer shown in SEQ ID NO. 294; the upstream primer shown in SEQ ID NO. 295 and the downstream primer shown in SEQ ID NO. 306; the upstream primer shown in SEQ ID NO. 307 and the downstream primer shown in SEQ ID NO. 308; the upstream primer shown in SEQ ID NO. 309 and the downstream primer shown in SEQ ID NO. 310; the upstream ... The upstream primer shown in SEQ ID NO. 311 and the downstream primer with the sequence shown in SEQ ID NO. 312; the upstream primer with the sequence shown in SEQ ID NO. 313 and the downstream primer with the sequence shown in SEQ ID NO. 314; the upstream primer with the sequence shown in SEQ ID NO. 315 and the downstream primer with the sequence shown in SEQ ID NO. 316; the upstream primer with the sequence shown in SEQ ID NO. 317 and the downstream primer with the sequence shown in SEQ ID NO. 318; and the sequence shown in SEQ ID NO.The upstream primer shown in SEQ ID NO. 319 and the downstream primer with the sequence shown in SEQ ID NO. 320; the upstream primer with the sequence shown in SEQ ID NO. 321 and the downstream primer with the sequence shown in SEQ ID NO. 322; the upstream primer with the sequence shown in SEQ ID NO. 323 and the downstream primer with the sequence shown in SEQ ID NO. 324; the upstream primer with the sequence shown in SEQ ID NO. 325 and the downstream primer with the sequence shown in SEQ ID NO. 326; the upstream primer with the sequence shown in SEQ ID NO. 327 and the downstream primer with the sequence shown in SEQ ID NO. 328; the upstream primer with the sequence shown in SEQ ID NO. 329 and the downstream primer with the sequence shown in SEQ ID NO. 330; the upstream primer with the sequence shown in SEQ ID NO. 331 and the downstream primer with the sequence shown in SEQ ID NO. 332; the upstream primer with the sequence shown in SEQ ID NO. 333 and the downstream primer with the sequence shown in SEQ ID NO. 334; the upstream primer with the sequence shown in SEQ ID NO. 335 and the downstream primer with the sequence shown in SEQ ID NO. 329; the upstream ...29; the downstream primer with the sequence shown in SEQ ID NO. 320; the upstream primer with the sequence shown in SEQ ID NO. 321 and the downstream primer with the sequence shown in SEQ The following primers are listed: the downstream primer shown in SEQ ID NO. 336; the upstream primer shown in SEQ ID NO. 337 and the downstream primer shown in SEQ ID NO. 338; the upstream primer shown in SEQ ID NO. 339 and the downstream primer shown in SEQ ID NO. 340; the upstream primer shown in SEQ ID NO. 341 and the downstream primer shown in SEQ ID NO. 342; the upstream primer shown in SEQ ID NO. 343 and the downstream primer shown in SEQ ID NO. 344; the upstream primer shown in SEQ ID NO. 345 and the downstream primer shown in SEQ ID NO. 346; the upstream primer shown in SEQ ID NO. 347 and the downstream primer shown in SEQ ID NO. 348; the upstream primer shown in SEQ ID NO. 349 and the downstream primer shown in SEQ ID NO. 350; the upstream primer shown in SEQ ID NO. 351 and the downstream primer shown in SEQ ID NO. 352; the upstream primer shown in SEQ ID NO. 346 and the downstream ... The upstream primer shown in NO. 353 and the downstream primer shown in SEQ ID NO. 354 are as follows;

[0023] The primer pair for detecting TSHR gene DNA mutations is selected from at least one of the following: the upstream primer with the sequence shown in SEQ ID NO. 405 and the downstream primer with the sequence shown in SEQ ID NO. 406; the upstream primer with the sequence shown in SEQ ID NO. 407 and the downstream primer with the sequence shown in SEQ ID NO. 408; the upstream primer with the sequence shown in SEQ ID NO. 409 and the downstream primer with the sequence shown in SEQ ID NO. 410; the upstream primer with the sequence shown in SEQ ID NO. 411 and the downstream primer with the sequence shown in SEQ ID NO. 412; the upstream primer with the sequence shown in SEQ ID NO. 413 and the downstream primer with the sequence shown in SEQ ID NO. 414; the upstream primer with the sequence shown in SEQ ID NO. 415 and the downstream primer with the sequence shown in SEQ ID NO. 416; the upstream primer with the sequence shown in SEQ ID NO. 417 and the downstream primer with the sequence shown in SEQ ID NO. 418; the upstream primer with the sequence shown in SEQ ID NO. 419 and the downstream primer with the sequence shown in SEQ ID NO. 420; the primer with the sequence shown in SEQ ID NO. 419; the primer with the sequence shown in SEQ ID NO. 419; the primer with the sequence shown in SEQ ID NO. 410; the primer with the sequence shown in SEQ ID NO. 410; the primer with the sequence shown in SEQ ID NO. 411; the primer with the sequence shown in SEQ ID NO. 412; the primer with the sequence shown in SEQ ID NO. 413; the primer with the sequence shown in SEQ ID NO. 414; the primer with the sequence shown in SEQ ID NO. 415; the primer with the sequence shown in SEQ ID NO. 416; the primer with the sequence shown in SEQ ID NO. 417 and the downstream primer with the sequence shown in SEQ ID NO. The upstream primer shown in SEQ ID NO. 421 and the downstream primer with the sequence shown in SEQ ID NO. 422; the upstream primer with the sequence shown in SEQ ID NO. 423 and the downstream primer with the sequence shown in SEQ ID NO. 424; the upstream primer with the sequence shown in SEQ ID NO. 425 and the downstream primer with the sequence shown in SEQ ID NO. 426; the upstream primer with the sequence shown in SEQ ID NO. 427 and the downstream primer with the sequence shown in SEQ ID NO. 428; the upstream primer with the sequence shown in SEQ ID NO. 429 and the downstream primer with the sequence shown in SEQ ID NO. 430; the upstream primer with the sequence shown in SEQ ID NO. 431 and the downstream primer with the sequence shown in SEQ ID NO. 432; the upstream primer with the sequence shown in SEQ ID NO. 433 and the downstream primer with the sequence shown in SEQ ID NO. 434; the upstream primer with the sequence shown in SEQ ID NO. 435 and the downstream primer with the sequence shown in SEQ ID NO. 436; the upstream primer with the sequence shown in SEQ ID NO. 437 and the downstream primer with the sequence shown in SEQ ID NO. 422; the upstream primer with the sequence shown in SEQ ID NO. 423 and the downstream primer with the sequence shown in SEQ ID NO. 424; the upstream primer with the sequence shown in SEQ ID NO. 425 and the downstream primer with the sequence shown in SEQ ID NO. 426; the upstream primer with the sequence shown in SEQ ID NO. 427 and the downstream primer with the sequence shown in SEQ ID NO. 428; the upstream primer with the sequence shown in SEQ ID NO. 429 and the downstream primer with the sequence shown in SEQ ID NO. 430; the upstream primer with the sequence shown in SEQ ID NO. 431 and the downstream primer with the sequence shown in SEQ ID NO. 432; the upstream primer with the sequence shown in SEQ The downstream primer shown in SEQ ID NO. 438; the upstream primer shown in SEQ ID NO. 439 and the downstream primer shown in SEQ ID NO. 440; the upstream primer shown in SEQ ID NO. 441 and the downstream primer shown in SEQ ID NO. 442; the upstream primer shown in SEQ ID NO. 443 and the downstream primer shown in SEQ ID NO. 444; and the primer shown in SEQ ID NO.The upstream primer shown in SEQ ID NO. 445 and the downstream primer with the sequence shown in SEQ ID NO. 446; the upstream primer with the sequence shown in SEQ ID NO. 447 and the downstream primer with the sequence shown in SEQ ID NO. 448; the upstream primer with the sequence shown in SEQ ID NO. 449 and the downstream primer with the sequence shown in SEQ ID NO. 450; the upstream primer with the sequence shown in SEQ ID NO. 451 and the downstream primer with the sequence shown in SEQ ID NO. 452; the upstream primer with the sequence shown in SEQ ID NO. 453 and the downstream primer with the sequence shown in SEQ ID NO. 454; the upstream primer with the sequence shown in SEQ ID NO. 455 and the downstream primer with the sequence shown in SEQ ID NO. 456; the upstream primer with the sequence shown in SEQ ID NO. 457 and the downstream primer with the sequence shown in SEQ ID NO. 458; the upstream primer with the sequence shown in SEQ ID NO. 459 and the downstream primer with the sequence shown in SEQ ID NO. 460; the upstream primer with the sequence shown in SEQ ID NO. 461 and the downstream primer with the sequence shown in SEQ ID NO. 445. The following primers are listed: downstream primer as shown in SEQ ID NO. 462; upstream primer as shown in SEQ ID NO. 463 and downstream primer as shown in SEQ ID NO. 464; upstream primer as shown in SEQ ID NO. 465 and downstream primer as shown in SEQ ID NO. 466; upstream primer as shown in SEQ ID NO. 467 and downstream primer as shown in SEQ ID NO. 468; upstream primer as shown in SEQ ID NO. 469 and downstream primer as shown in SEQ ID NO. 470; upstream primer as shown in SEQ ID NO. 471 and downstream primer as shown in SEQ ID NO. 472.

[0024] The primer pair for detecting ARAF gene DNA mutations includes: an upstream primer with the sequence shown in SEQ ID NO.3 and a downstream primer with the sequence shown in SEQ ID NO.4;

[0025] The primer pair for detecting AKT1 gene DNA mutations includes: an upstream primer with the sequence shown in SEQ ID NO.1 and a downstream primer with the sequence shown in SEQ ID NO.2;

[0026] The primer pair for detecting CTNNB1 gene DNA mutations is selected from at least one of the following: the upstream primer with the sequence shown in SEQ ID NO. 33 and the downstream primer with the sequence shown in SEQ ID NO. 34; the upstream primer with the sequence shown in SEQ ID NO. 35 and the downstream primer with the sequence shown in SEQ ID NO. 36; the upstream primer with the sequence shown in SEQ ID NO. 37 and the downstream primer with the sequence shown in SEQ ID NO. 38; the upstream primer with the sequence shown in SEQ ID NO. 39 and the downstream primer with the sequence shown in SEQ ID NO. 40;

[0027] The primer pair for detecting RAF1 gene DNA mutations includes: an upstream primer with a sequence as shown in SEQ ID NO.355 and a downstream primer with a sequence as shown in SEQ ID NO.356.

[0028] In some embodiments, the primer composition further includes primer pairs for detecting RNA fusion variants of the ALK, BRAF, NTRK1, NTRK2, NTRK3, PPARG, RET, and THADA genes;

[0029] The ALK gene RNA fusion variant includes RNA fusion variants of the ALK gene and EML4, KIF5B or TPM3 genes, and the primer pair includes: upstream primers with sequences as shown in SEQ ID NO. 551-556, SEQ ID NO. 561-566, SEQ ID NO. 581-584 and downstream primers with sequences as shown in SEQ ID NO. 473-480;

[0030] The BRAF gene RNA fusion mutation is an RNA fusion mutation of the BRAF gene and the AKAP9 gene, and the primer pair includes: an upstream primer with a sequence as shown in SEQ ID NO. 485-490 and a downstream primer with a sequence as shown in SEQ ID NO. 481-484 and SEQ ID NO. 589-590;

[0031] The NTRK1 gene RNA fusion variant is an RNA fusion variant of the NTRK1 gene and the TPM3 gene, and the primer pair includes: an upstream primer with a sequence as shown in SEQ ID NO. 581-584 and a downstream primer with a sequence as shown in SEQ ID NO. 491-508;

[0032] The NTRK2 gene RNA fusion variant is an RNA fusion variant of the NTRK2 gene and the BCR gene, and the primer pair includes: an upstream primer with a sequence as shown in SEQ ID NO. 591-592 and a downstream primer with a sequence as shown in SEQ ID NO. 509-518;

[0033] The NTRK3 gene RNA fusion variant is an RNA fusion variant of the NTRK3 gene and the ETV6 gene. The primer pair includes: an upstream primer with sequences as shown in SEQ ID NO. 523-530 and SEQ ID NO. 557-560 and a downstream primer with sequences as shown in SEQ ID NO. 519-522 and SEQ ID NO. 531-532.

[0034] The PPARG gene RNA fusion variant is an RNA fusion variant of the PPARG gene and the PAX8 gene, and the primer pair includes: an upstream primer with sequences shown in SEQ ID NO. 573-578 and a downstream primer with sequences shown in SEQ ID NO. 587-588;

[0035] The RET gene RNA fusion variant includes RNA fusion variants of the RET gene and the KIF5B, CCDC6, NCOA4, or PRKAR1A genes, and the primer pair includes: upstream primers with sequences as shown in SEQ ID NO. 561-566, SEQ ID NO. 545-550, SEQ ID NO. 567-572, and SEQ ID NO. 579-580, and downstream primers with sequences as shown in SEQ ID NO. 533-538;

[0036] The THADA gene RNA fusion variant is an RNA fusion variant of the THADA gene and the IGF2BP3 gene. The primer pair includes an upstream primer with a sequence as shown in SEQ ID NO. 539-544 and a downstream primer with a sequence as shown in SEQ ID NO. 585-586.

[0037] In some embodiments, the primer composition includes primer pairs for detecting DNA mutations in the BRAF, KRAS, NRAS, TERT, TP53, RET, CDKN2A, DICER1, EIF1AX, GNAS, HRAS, PIK3CA, PTEN, TSHR, ARAF, AKT1, CTNNB1, and RAF1 genes, and primer pairs for detecting RNA fusion variations in the ALK, BRAF, NTRK1, NTRK2, NTRK3, PPARG, RET, and THADA genes.

[0038] In some embodiments, at least 50% of the upstream primers in the primer composition have a nucleotide fragment with the sequence shown in SEQ ID NO. 593 attached to their 5' ends, and the corresponding downstream primers have a nucleotide fragment with the sequence shown in SEQ ID NO. 594 attached to their 3' ends.

[0039] Preferably, the upstream primer for detecting DNA mutations in the AKT1, ARAF, BRAF, CDKN2A, CTNNB1, DICER1, and GNAS genes, and for detecting RNA fusion variations in the BRAF gene, has a nucleotide fragment with the sequence shown in SEQ ID NO. 593 attached to its 5' end, and the corresponding downstream primer has a nucleotide fragment with the sequence shown in SEQ ID NO. 594 attached to its 3' end.

[0040] A second aspect of the invention provides the use of the primer composition described above in the preparation of products for detecting benign or malignant gene mutations associated with said thyroid nodules.

[0041] In some embodiments, the product is a reagent kit.

[0042] A third aspect of the present invention provides a kit for detecting gene mutations related to benign or malignant thyroid nodules, the kit comprising the primer composition described above.

[0043] A fourth aspect of the present invention provides a method for detecting gene mutations related to benign or malignant thyroid nodules, comprising the following steps: extracting DNA and RNA from the sample to be tested, reverse transcribing the RNA to obtain cDNA; using the DNA and cDNA as templates, performing multiplex PCR amplification using the primer composition described above or the kit described above, constructing a library, and sequencing.

[0044] A fifth aspect of the present invention provides a system for detecting gene mutations related to benign or malignant thyroid nodules, comprising:

[0045] Detection module: The detection module includes a multiplex amplification module for genes related to benign and malignant thyroid nodules, a library construction module, and a sequencing module; the multiplex amplification module for genes related to benign and malignant thyroid nodules includes the primer composition described above or the kit described above;

[0046] Data analysis module: The data analysis module analyzes the sequencing results of the sequencing module, including filtering data below the quality control threshold, removing adapters, aligning sequences to the corresponding positions in the reference genome data, annotating gene mutations, and performing statistical analysis of the corresponding data.

[0047] In some embodiments, the sequencing module is an Illumina sequencing module.

[0048] This invention provides a primer composition for detecting DNA mutations (including single nucleotide variants (SNVs) and small fragment insertions and deletions (Indels)) and RNA fusion variants in genes related to benign and malignant thyroid nodules. The primer pairs in the primer composition can be amplified individually, and can also be amplified efficiently, with high sensitivity and high specificity, under the same PCR reaction system and reaction procedure for the test DNA and cDNA template. There is no significant interference between the primers. A single multiplex PCR amplification can rapidly and accurately amplify the specific nucleic acid sequences of multiple regions of genes related to benign and malignant thyroid nodules. Subsequently, by constructing a library and sequencing, the gene mutation status of the relevant regions can be obtained, assisting in the clinical diagnosis of benign and malignant thyroid nodules.

[0049] Furthermore, in this invention, specific nucleotide sequences are respectively ligated to the 5' end of the upstream primer and the 3' end of the corresponding downstream primer in appropriate proportions in the primer composition. These nucleotide sequences do not affect the amplification effect of the primers in the primer composition and enable non-specific amplification fragments in the multiplex PCR amplification system to form a "neck loop" structure, preventing them from serving as templates for further amplification. This reduces non-specific amplification in the multiplex PCR amplification system, further improving the uniformity and specificity of multiplex PCR amplification. It effectively reduces the amount of nucleic acid input required for detection by the primer composition of this invention and lowers the limit of detection for mutation frequency (down to 0.5%), while also increasing the number of reads detected. This allows for a nucleic acid input as low as 1 ng for samples with a mutation frequency of 2% or higher, and as low as 5 ng for samples with 1% mutation frequency. Therefore, the primer composition of this invention is highly suitable for detecting related gene mutations in biological samples with small sample volumes and low nucleic acid concentrations, including thyroid fine-needle aspiration samples.

[0050] The detection method of this invention can simultaneously detect gene variations at the DNA and RNA levels. It is simple to operate, low in cost, and can shorten the detection time to 4 days, showing broad application prospects. Attached Figure Description

[0051] Figure 1 This is a flowchart of the detection method of the present invention. Detailed Implementation

[0052] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0053] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in the fourth edition of *Molecular Cloning: A Laboratory Manual*, edited by Green and Sambrook, published in 2013, or according to the manufacturer's recommendations. All commonly used chemical reagents used in the examples are commercially available products.

[0054] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0055] Furthermore, as used herein, the term "or" is an inclusive "or" sign and is equivalent to the term "and / or" unless the context clearly specifies otherwise. The term "based on" is not exclusive and allows for basing on other factors not described unless the context clearly specifies otherwise. Additionally, throughout the specification, the meanings of "an," "a," and "the" include plural indicators. The meaning of "in" includes both "in" and "on."

[0056] The present invention will be further described in detail below with reference to specific embodiments.

[0057] Example 1

[0058] This embodiment provides a primer composition, kit, and method for detecting gene mutations related to benign and malignant thyroid nodules.

[0059] 1. Primer composition

[0060] The primer composition includes primers for detecting DNA mutations (including single nucleotide variants, small fragment insertions and deletions) and RNA fusion variants of genes related to benign and malignant thyroid nodules. The related genes and representative variants are shown in Table 1.

[0061] Table 1

[0062]

[0063]

[0064] The sequence information of the primers for detecting DNA mutations in genes related to benign and malignant thyroid nodules and the location information of the amplified regions in the genome are shown in Table 2. The version number of the genome being compared is hg19.

[0065] Table 2 Primers for DNA mutation detection

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077] RNA fusion mutations occur in two genes, and the two genes involved in the fusion are not fixed. Detection primers for fusion mutations include upstream primers (for the upstream fusion gene) and downstream primers (for the downstream fusion gene). As shown in Table 1, the ALK gene can undergo RNA fusion mutations with the EML4, KIF5B, or TPM3 genes; the BRAF gene can undergo RNA fusion mutations with the AKAP9 gene; the NTRK1 gene can undergo RNA fusion mutations with the TPM3 gene; the NTRK2 gene can undergo RNA fusion mutations with the BCR gene; the NTRK3 gene can undergo RNA fusion mutations with the ETV6 gene; the PPARG gene can undergo RNA fusion mutations with the PAX8 gene; the RET gene can undergo RNA fusion mutations with the KIF5B, CCDC6, NCOA4, or PRKAR1A genes; and the THADA gene can undergo RNA fusion mutations with the IGF2BP3 gene. The primers for detecting fusion mutations in this invention are designed at the fusion sites (fusion sites) of the above genes, and two primers are designed for the same fusion site to ensure detection specificity (labeled as primer 1 and primer 2 in Table 3 below). A gene can have multiple fusion sites, and a gene can be either an upstream gene (the amplification primer is the upstream primer) or a downstream gene (the amplification primer is the downstream primer).

[0078] The sequence information of the primers for detecting relevant RNA fusion variants, the location information of the amplified region in the genome, and the upstream and downstream information of the primers are shown in Table 3. The amplified region corresponding to the primer for detecting RNA fusion variants is the location information of the corresponding transcript in the genome, and the version number of the genome being compared is hg19.

[0079] In Table 3, “Gene Name-F” indicates that the gene is located upstream of the fusion variant, and an upstream primer (Forward Primer) was designed for its fusion position; “Gene Name-R” indicates that the gene is located downstream of the fusion variant, and a downstream primer (Reverse Primer) was designed for its fusion position.

[0080] Table 3 Primers for RNA fusion variant detection

[0081]

[0082]

[0083]

[0084]

[0085] The inventors further discovered that for primers in Tables 2 and 3, when the 5' end linking sequence of the upstream primer is the nucleotide fragment shown in SEQ ID NO. 593 for 50%–100% (including 50%–60%, 60%–70%, 70%–80%, 80%–90%, 90%–100%, 50%–70%, 50%–80%, 50%–90%, 60%–80%, 60%–90%, 60%–100%, 70%–90%, 70%–100%, 80%–100%), and the 3' end linking sequence of the corresponding downstream primer is the nucleotide fragment shown in SEQ ID NO. 594, it does not affect the amplification effect of the primers, and can effectively reduce non-specific amplification in the multiplex PCR amplification system, further improving the uniformity and specificity of multiplex PCR amplification. In this embodiment, the upstream primer (approximately 51%) for mutation detection of the gene shown in bold and underlined has a nucleotide fragment with the sequence shown in SEQ ID NO. 593 attached to its 5' end, and the corresponding downstream primer has a nucleotide fragment with the sequence shown in SEQ ID NO. 594 attached to its 3' end.

[0086] SEQ ID NO. 593: TAGGTTTTACCCAT; SEQ ID NO. 594: CGATATTTTGTGG.

[0087] 2. Reagent kit

[0088] The primers described in Tables 2 and 3 are mixed evenly to obtain a primer composition. The final concentration range of each primer in the primer composition is 0.5-0.7 μM, and in this embodiment, it is preferably 0.6 μM. The kit contains the primer composition.

[0089] In addition to the primer composition, the kit may also include other components required for the detection. For example, in addition to the primer composition, other components required for the detection are purchased from Shanghai Zhengu Biotechnology Co., Ltd.'s Thyroid Cancer Multigene Detection Kit [KY], catalog number CT0470. The kits containing the reagents required for the detection are packaged into kits 1 to 5, and the specific components are shown in Table 4.

[0090] Table 4

[0091]

[0092]

[0093] 3. Detection Method

[0094] The detection method includes the following steps:

[0095] Step 1, Sample Preparation: Nucleic acid extraction (DNA + RNA) is performed on the thyroid fine-needle aspiration sample;

[0096] Step 2: Reverse transcribe the RNA in the sample into cDNA, and use it together with the original DNA in the sample as a template for subsequent multiplex PCR amplification;

[0097] Step 3: Perform multiplex PCR amplification to obtain one round of amplicon products, purify the excess primers in the system, ligate index tags to both ends of the amplicon products to complete the amplicon library construction, perform library quality control, and then sequence the amplicon.

[0098] Step 4, Sequencing: Sequencing was performed using an Illumina NovaSeq 6000 sequencer (PE150).

[0099] Step 5: Bioinformatics analysis: Compare the amplicon sequencing results with the human reference genome (version number hg19) to determine the mutation status of the sample genes.

[0100] Figure 1 This is a flowchart of the detection method of the present invention.

[0101] The specific operation steps of the detection method are as follows: detection is performed using the reagent kit components shown in Table 4.

[0102] 1. cDNA synthesis

[0103] 1.1 Take the components from kit 1 for the experiment. Take samples according to the amount of RNA input 20-100ng and DNA input 5-60ng. Take 0.2ml PCR tubes according to the number of samples and mark the sample number on the tube cap.

[0104] 1.2 Add the components in the order shown in Table 5 below and prepare the reaction system on an ice box. Since the extracted nucleic acid sample includes both DNA and RNA, the addition of RNA in Table 5 will carry a corresponding volume of DNA. This will not affect the reverse transcription reaction. The DNA added in the subsequent multiplex PCR amplification reaction system (Table 7) is the DNA added again. The sum of the amount of DNA added in Table 7 and the amount of DNA added in Table 5 is the total amount of DNA input for detection.

[0105] Table 5

[0106] Components Volume (μL) Proto-nucleic acid (DNA+RNA) 7 Reagent 1 2.5 Reagent 2 0.5 Total volume 10

[0107] Reagent 1 and Reagent 2 can be premixed on an ice plate.

[0108] 1.3 Vortex and centrifuge the mixture in the PCR tube, then place it on a PCR instrument to perform the reactions shown in Table 6 below:

[0109] Table 6

[0110] temperature reaction time Cycle number 50℃ 15min 1 85℃ 5s 1 8℃ Hold 1

[0111] 2. Purification of cDNA after synthesis

[0112] 2.1 Preparation before purification: Balancing magnetic beads (reagent 11): Remove the Agencourt AMPure XP Beads from 4°C and place them at room temperature for at least 30 minutes. The temperature of the AMPure beads will affect the purification effect.

[0113] 2.2 cDNA product purification:

[0114] 2.2.1 Mixing magnetic beads: Shake the AMPure XP beads until they are mixed evenly (Important note: Make sure to mix the AMPure beads evenly before purification, otherwise it will affect the purification effect).

[0115] 2.2.2 Adding magnetic beads: Add 30 μL (3 times the volume) of magnetic beads to each well. Use a pipette to pipette 10 times. If air bubbles are generated at the bottom of the tube during pipetting, briefly centrifuge and remix.

[0116] 2.2.3 Product binding: Incubate the reaction plate / tube at room temperature for 5 min to allow the PCR products to bind to the magnetic beads.

[0117] 2.2.4 Separating magnetic beads: Place the PCR plate / tube on a magnetic rack for 5 minutes, until the solution becomes clear.

[0118] 2.2.5 Remove supernatant: Carefully remove the supernatant from each tube, being careful not to touch or blow away the magnetic beads.

[0119] 2.2.6 Cleaning the magnetic beads: Keep the PCR plate / tube on the magnetic rack. Add 150 μL of freshly prepared 70% ethanol to each tube. Incubate for 30 seconds, then aspirate all supernatant. Be careful not to touch the magnetic beads.

[0120] Important Note: Avoid contact between 70% ethanol and air. Otherwise, the ethanol concentration will change, affecting the cleaning effect on the magnetic beads. Prepare only enough 70% ethanol for each use.

[0121] 2.2.7 Second Wash: Repeat the previous step. The remaining 70% ethanol can be used for the purification of the second round of PCR products.

[0122] 2.2.8 Remove residual ethanol: Completely remove any remaining trace amounts of ethanol from each tube. Briefly centrifuge for 10-15 seconds, place the PCR plate / tube back on the magnetic rack, and use a 10-20 μL pipette tip to remove any remaining ethanol solution from the bottom of the tube.

[0123] 2.2.9 Drying the magnetic beads: Keep the PCR reaction plate on the magnetic rack and let it sit at room temperature for 2-5 minutes.

[0124] Important Note: Do not allow the magnetic beads to become overly dry. Small cracks appearing in the middle of the bead cluster indicate that the beads are sufficiently dry. If large cracks appear running through the entire cluster, or if the beads break into small, thin pieces, they are overly dry. Overly dry magnetic beads will be difficult to resuspend.

[0125] 2.2.10 Resuspending the magnetic beads: Remove the PCR plate / tube from the magnetic rack and immediately add 12 μL of nuclease-free water to each tube to resuspend the magnetic beads. Gently pipette the suspension 10 times. If air bubbles appear at the bottom of the tube, briefly centrifuge and remix.

[0126] 2.2.11 Elution: Place the PCR plate / tube at room temperature for 5 minutes to fully elute the PCR products.

[0127] 2.2.12 Separate the supernatant: Place the PCR plate / tube back into the magnetic rack until the solution becomes clear, which may take up to 5 minutes. Transfer 10 μL of the supernatant to a new PCR tube (pause point: can be stored at -20℃) to obtain the cDNA product.

[0128] 3. GS-PCR amplification (multiplex PCR reaction)

[0129] 3.1 Take the components from kit 2 for the experiment. Take 0.2 ml PCR tubes according to the number of samples and label the tube caps with the sample numbers.

[0130] 3.2 Add the components in the order shown in Table 7 below to prepare the reaction system on an ice box:

[0131] Table 7

[0132] Components Volume (μL) Reagent 4 25 Reagent 5 6 Reagent 6 2.5 cDNA product obtained in step 2 10 Sample DNA X Reagent 3 6.5-X Total volume 50

[0133] Reagents 4, 5, and 6 can be premixed on an ice plate.

[0134] 3.3 Vortex and centrifuge the mixture in the PCR tube, then place it on a PCR instrument to perform the reactions shown in Table 8 below:

[0135] Table 8

[0136]

[0137] Gene-specific PCR products were obtained.

[0138] 4. Enzymatic digestion of gene-specific PCR primers

[0139] 4.1 Preparation: Take out reagent 8 from kit 3 and place it on ice. Store gene-specific PCR products at room temperature.

[0140] 4.2 Dilute Reagent 8. Dilute Reagent 8 with nuclease-free water at a ratio of 3:2 according to the number of samples. After preparing the reaction solution, place it on ice. Prepare the enzymatic digestion system according to the reaction system in Table 9 below:

[0141] Table 9

[0142] Components Volume (μL) Reagent 7 (after dilution) 5 The gene-specific PCR product obtained in step 3 50 Total volume 55

[0143] 4.3 Vortex and centrifuge the mixture in the PCR tube, then place it on a PCR instrument to perform the reactions shown in Table 10 below:

[0144] Table 10

[0145] temperature reaction time Cycle number 37℃ 20min 1 80℃ 10min 1 8℃ Hold 1

[0146] Gene-specific PCR products were obtained after enzymatic digestion and purification.

[0147] 5. Purification of gene-specific PCR products

[0148] 5.1 Preparation before purification: Balancing magnetic beads (reagent 11): Remove the Agencourt AMPure XP Beads from 4°C and place them at room temperature for at least 30 minutes. The temperature of the AMPure beads will affect the purification effect.

[0149] 5.2 Purification of gene-specific PCR products:

[0150] 5.2.1 Mixing magnetic beads: Shake the AMPure XP beads until they are evenly mixed (Important note: Make sure to mix the AMPure beads evenly before purification, otherwise it will affect the purification effect).

[0151] 5.2.2 Adding magnetic beads: Add 55 μL (1.0 volume) of magnetic beads to each well. Use a pipette to pipette 10 times. If air bubbles are generated at the bottom of the tube during pipetting, briefly centrifuge and remix.

[0152] 5.2.3 Product binding: Incubate the reaction plate / tube at room temperature for 5 min to allow the pCR product to bind to the magnetic beads.

[0153] 5.2.4 Separating magnetic beads: Place the PCR plate / tube on a magnetic rack for 5 minutes, until the solution becomes clear.

[0154] 5.2.5 Remove supernatant: Carefully remove the supernatant from each tube, being careful not to touch or blow away the magnetic beads.

[0155] 5.2.6 Cleaning the magnetic beads: Keep the PCR plate / tube on the magnetic rack. Add 150 μL of freshly prepared 70% ethanol to each tube. Incubate for 30 seconds, then aspirate all supernatant. Be careful not to touch the magnetic beads.

[0156] Important Note: Avoid contact between 70% ethanol and air. Otherwise, the ethanol concentration will change, affecting the cleaning effect on the magnetic beads. Prepare only enough 70% ethanol for each use.

[0157] 5.2.7 Second Wash: Repeat the previous step. The remaining 70% ethanol can be used for the purification of the second round of PCR products.

[0158] 5.2.8 Remove residual ethanol: Completely remove any remaining trace amounts of ethanol from each tube. Briefly centrifuge for 10-15 seconds, place the PCR plate / tube back on the magnetic rack, and use a 10-20 μL pipette tip to remove any remaining ethanol solution from the bottom of the tube.

[0159] 5.2.9 Drying the magnetic beads: Keep the PCR reaction plate on the magnetic rack and let it sit at room temperature for 2-5 minutes.

[0160] Important Note: Do not allow the magnetic beads to become overly dry. Small cracks appearing in the middle of the bead cluster indicate that the beads are sufficiently dry. If large cracks appear running through the entire cluster, or if the beads break into small, thin pieces, they are overly dry. Overly dry magnetic beads will be difficult to resuspend.

[0161] 5.2.10 Resuspending the magnetic beads: Remove the PCR plate / tube from the magnetic rack and immediately add 20 μL of nuclease-free water to each tube to resuspend the magnetic beads. Gently pipette the suspension 10 times. If air bubbles appear at the bottom of the tube, briefly centrifuge and remix.

[0162] 5.2.11 Elution: Place the PCR plate / tube at room temperature for 5 minutes to fully elute the PCR products.

[0163] 5.2.12 Separate the supernatant: Place the PCR plate / tube back into the magnetic rack until the solution becomes clear. This process may take 5 minutes. Aspirate 18 μL of the supernatant into a new PCR tube (pause point: can be stored at -20℃) to obtain the gene-specific PCR purified product.

[0164] 6. Indexing PCR (Library Amplification)

[0165] 6.1 Reagent preparation: Take out reagents 8, 9 and 10 from kit 4 and place them on ice.

[0166] Prepare the reaction solution according to the system in Table 11 below:

[0167] Table 11

[0168] Components Volume (μL) Reagent 8 25 Reagent 9 4 Reagent 10 4 The gene-specific PCR purification product obtained in step 5 7 Reagent 3 10 Total volume 50

[0169] 6.2 Vortex and centrifuge the mixture in the PCR tube, then place it on a PCR instrument to perform the reactions shown in Table 12 below:

[0170] Table 12

[0171]

[0172] The amplicon library was obtained.

[0173] 7. Library purification:

[0174] 7.1 Preparation before purification: Equilibrium magnetic beads (reagent 11): Remove Agencourt AMPure XP Beads from 4°C and place them at room temperature for at least 30 minutes. The temperature of the AMPure beads will affect the purification effect.

[0175] 7.2 Library purification:

[0176] 7.2.1 Mixing magnetic beads: Shake the AMPure XP beads until they are mixed evenly (Important note: Make sure to mix the AMPure beads evenly before purification, otherwise it will affect the purification effect).

[0177] 7.2.2 Adding magnetic beads: Add 40 μL (0.8 times the volume) of magnetic beads to each well. Use a pipette to pipette 10 times. If air bubbles are generated at the bottom of the tube during pipetting, briefly centrifuge and remix.

[0178] 7.2.3 Product binding: Incubate the reaction plate / tube at room temperature for 5 min to allow the PCR products to bind to the magnetic beads.

[0179] 7.2.4 Separating magnetic beads: Place the PCR plate / tube on a magnetic rack for 5 minutes, until the solution becomes clear.

[0180] 7.2.5 Remove supernatant: Carefully remove the supernatant from each tube, being careful not to touch or blow away the magnetic beads.

[0181] 7.2.6 Cleaning the magnetic beads: Keep the PCR plate / tube on the magnetic rack. Add 150 μL of freshly prepared 70% ethanol to each tube. Incubate for 30 seconds, then aspirate all supernatant. Be careful not to touch the magnetic beads.

[0182] Important Note: Avoid contact between 70% ethanol and air. Otherwise, the ethanol concentration will change, affecting the cleaning effect on the magnetic beads. Prepare only enough 70% ethanol for each use.

[0183] 7.2.7 Second Wash: Repeat the previous step. The remaining 70% ethanol can be used for the purification of the second round of PCR products.

[0184] 7.2.8 Remove residual ethanol: Completely remove any remaining trace amounts of ethanol from each tube. Centrifuge briefly for 10-15 seconds, return the PCR plate / tube to the magnetic rack, and use a 10-20 μL pipette tip to remove any remaining ethanol solution from the bottom of the tube.

[0185] 7.2.9 Drying the magnetic beads: Keep the PCR reaction plate on the magnetic rack and let it sit at room temperature for 2-5 minutes.

[0186] Important Note: Do not allow the magnetic beads to become overly dry. Small cracks appearing in the middle of the bead cluster indicate that the beads are sufficiently dry. If large cracks appear running through the entire cluster, or if the beads break into small, thin pieces, they are overly dry. Overly dry magnetic beads will be difficult to resuspend.

[0187] 7.2.10 Resuspending the magnetic beads: Remove the PCR plate / tube from the magnetic rack and immediately add 22 μL of nuclease-free water to each tube to resuspend the magnetic beads. Gently pipette the suspension 10 times. If air bubbles appear at the bottom of the tube, briefly centrifuge and remix.

[0188] 7.2.11 Elution: Place the PCR plate / tube at room temperature for 5 minutes to fully elute the PCR products.

[0189] 7.2.12 Separate the supernatant: Place the PCR plate / tube back into the magnetic rack until the solution becomes clear. This process may take 5 minutes. Aspirate 20 μL of the supernatant into a new PCR tube (pause point: can be stored at -20℃) to obtain the purified amplicon library.

[0190] 8. Document Quality Inspection

[0191] 8.1 Qubit quantification: Take 1 μL of the library and use Qubit to detect the library concentration. The library quality control standard is: concentration greater than 1 ng / ul and total amount not less than 20 ng.

[0192] 8.2Qseq analysis of the library length showed that the main peak was around 400 bytes.

[0193] 9. Sequencing

[0194] Sequencing was performed using the Illumina sequencing platform and the NovaSeq 6000 sequencer. It is recommended to use the Novaseq6000S1Reagent Kit v1.5 (PE150) sequencing chip for sequencing, with 1GB of data per sample.

[0195] 10. Bioinformatics Automated Process Analysis and Data Quality Control

[0196] 10.1 Data analysis was performed using the bioinformatics software shown in Table 13 below:

[0197] Table 13

[0198] software Software version use Function Fastp v0.23.2 Cut Adapter To the connector Sentieon v202112.04 reads mapping Sequence alignment annovar v2020 SNV / Indel annotation Annotation SNV / Indel variants STAR-Fusion v1.10.0 Gene Fusion Calling Detecting gene fusion variants Arriba v2.3.0 Gene Fusion Calling Detecting gene fusion variants VarDict 1.8.3 SNV / Indel Calling Detection of SNV / Indel variants

[0199] 10.2 Write a rules program using Snakemake language rules. Use graphviz to generate a process flow diagram and use the -dry-run mode to check if the program logic is correct. After checking, run the program to execute no less than 1000 tasks, and check that the number of errors is less than 1.

[0200] 10.3 The quality control requirements for sequencing data are as follows: Taking into account the actual sample sequencing quality and the requirements for the mutation detection software to accurately detect mutations, the quality control threshold requirements for sequencing data are shown in Table 14 below.

[0201] Table 14

[0202] index illustrate Require Average sequencing depth (X) On average, each base was sequenced an average of [number] times. ≥3000X Base ratio >500x Percentage of bases sequenced more than 500 times ≥85％ Sequence alignment rate (On Target) The degree of matching between sequencing results and the reference genome ≥80％ Q30 The reliability of this base is 99.9%. ≥85％

[0203] Example 2

[0204] The detection accuracy of the detection kit of the present invention was verified, and the kit and detection method are the same as in Example 1.

[0205] 1. Verification of the accuracy of FFPE standard SNV / Indel: 200ng of FFPE standard was added for testing, and the accuracy comparison results are shown in Table 15 below.

[0206] Table 15

[0207]

[0208]

[0209] Sequencing tests were performed on the FFPE standard, and a total of 6 SNV mutation sites were detected within the detection range. The measured mutation frequency was basically consistent with the theoretical mutation frequency, which met the expected results.

[0210] 2. Verification of SNV / Indel accuracy of clinical samples with known gene mutation results (hybrid capture sequencing): 200 ng of DNA sample was used for testing, and the accuracy comparison results are shown in Table 16 below.

[0211] Table 16

[0212]

[0213]

[0214] Twenty-one clinical samples were tested, and a total of 25 SNV mutation sites were detected within the detection range. The measured mutation frequency was basically consistent with the original mutation frequency, which was in line with the expected results.

[0215] Example 3

[0216] The repeatability verification of the detection kit of the present invention is performed, and the kit and detection method are the same as in Example 1.

[0217] 1. By conducting batch-to-batch and intra-batch consistency tests on FFPE standard products (SNV / Indel) and statistically analyzing the results, the precision verification results are shown in Table 17 below.

[0218] Table 17

[0219]

[0220] Intra-batch consistency: One FFPE standard (GW-OGTM800) was repeated twice within the same batch, and the SNV / Indel were consistently detected, resulting in an intra-batch consistency of 100%.

[0221] Inter-batch consistency: One FFPE standard (GW-OGTM800) was repeated three times in different batches, and the SNV / Indel was consistently detected in all three batches, with an inter-batch consistency of 100%.

[0222] The results show that the kit of the present invention has excellent detection repeatability.

[0223] Example 4

[0224] The detection kit of this invention verifies the minimum amount of nucleic acid input, and the kit and detection method are the same as in Example 1.

[0225] 1. The limit of detection was set for FFPE standard (GW-OGTM800) with different DNA input amounts (5ng, 10ng, 25ng, 50ng, 100ng). The results are shown in Table 18 below.

[0226] Table 18

[0227]

[0228] Sequencing tests were performed on FFPE standards with different input amounts. Under five DNA input amounts (5ng, 10ng, 25ng, 50ng, and 100ng), two 1% SNV sites could be stably detected. Therefore, the detection kit of this invention can still stably detect 1% of SNV sites even with a DNA input amount as low as 5ng.

[0229] Further testing was conducted with even lower DNA input levels.

[0230] The limit of detection was set for FFPE standard (GW-OGTM800) with different DNA input amounts (3ng, 2ng, 1ng), and the detection results are shown in Table 19 below.

[0231] Table 19

[0232]

[0233] The results showed that even with lower DNA input levels (3ng, 2ng, 1ng) for FFPE standards, more than 2% of SNV sites could still be stably detected even with a DNA input level as low as 1ng.

[0234] Example 5

[0235] The detection kit of the present invention verifies the detection limit of the lowest detection limit. The kit and detection method are the same as in Example 1.

[0236] 1. The 5% mutation frequency of the ctDNA standard (GW-OCTM001) was diluted to a mutation frequency of 0.5%, and 100 ng was added to verify the limit of detection of mutation frequency. The results are shown in Table 20 below.

[0237] Table 20

[0238]

[0239]

[0240] The limit of detection was validated for ctDNA standards with a mutation frequency of 0.5%. A total of 11 SNV mutation sites were detected within the detection range, and the measured mutation frequencies all fell within the theoretical mutation frequency range. Therefore, the minimum detectable mutation frequency of this kit can be as low as 0.5%.

[0241] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A primer composition for detecting gene mutations related to benign and malignant thyroid nodules, characterized in that, Primer pairs for detecting DNA mutations in the BRAF, KRAS, NRAS, TERT, TP53, RET, CDKN2A, DICER1, EIF1AX, GNAS, HRAS, PIK3CA, PTEN, TSHR, ARAF, AKT1, CTNNB1, and RAF1 genes, and primer pairs for detecting RNA fusion variations in the ALK, BRAF, NTRK1, NTRK2, NTRK3, PPARG, RET, and THADA genes. The primer pairs for detecting BRAF gene DNA mutations include: an upstream primer with the sequence shown in SEQ ID NO. 5 and a downstream primer with the sequence shown in SEQ ID NO. 6; an upstream primer with the sequence shown in SEQ ID NO. 7 and a downstream primer with the sequence shown in SEQ ID NO. 8; and an upstream primer with the sequence shown in SEQ ID NO. 9 and a downstream primer with the sequence shown in SEQ ID NO.

10. The primer pairs for detecting KRAS gene DNA mutations include: an upstream primer with the sequence shown in SEQ ID NO. 121 and a downstream primer with the sequence shown in SEQ ID NO. 122; an upstream primer with the sequence shown in SEQ ID NO. 123 and a downstream primer with the sequence shown in SEQ ID NO. 124; an upstream primer with the sequence shown in SEQ ID NO. 125 and a downstream primer with the sequence shown in SEQ ID NO. 126; and an upstream primer with the sequence shown in SEQ ID NO. 127 and a downstream primer with the sequence shown in SEQ ID NO.

128. The primer pairs for detecting NRAS gene DNA mutations include: an upstream primer with the sequence shown in SEQ ID NO. 129 and a downstream primer with the sequence shown in SEQ ID NO. 130; an upstream primer with the sequence shown in SEQ ID NO. 131 and a downstream primer with the sequence shown in SEQ ID NO. 132; an upstream primer with the sequence shown in SEQ ID NO. 133 and a downstream primer with the sequence shown in SEQ ID NO. 134; and an upstream primer with the sequence shown in SEQ ID NO. 135 and a downstream primer with the sequence shown in SEQ ID NO.

136. The primer pair for detecting TERT gene DNA mutations includes: an upstream primer with the sequence shown in SEQ ID NO. 385 and a downstream primer with the sequence shown in SEQ ID NO. 386; The primer pairs for detecting TP53 gene DNA mutations include: the upstream primer and the downstream primer shown in SEQ ID NO. 387; the upstream primer and the downstream primer shown in SEQ ID NO. 388; the upstream primer and the downstream primer shown in SEQ ID NO. 390; the upstream primer and the downstream primer shown in SEQ ID NO. 391; the upstream primer and the downstream primer shown in SEQ ID NO. 392; the upstream primer and the downstream primer shown in SEQ ID NO. 393; the upstream primer and the downstream primer shown in SEQ ID NO. 394; the upstream primer and the downstream primer shown in SEQ ID NO. 395; the upstream primer and the downstream primer shown in SEQ ID NO. 396; the upstream primer and the downstream primer shown in SEQ ID NO. 397; the upstream primer and the downstream primer shown in SEQ ID NO. 398; the upstream primer and the downstream primer shown in SEQ ID NO. 399; and the upstream primer and the downstream primer shown in SEQ ID NO. 400; The downstream primer shown in SEQ ID NO. 402; the upstream primer with the sequence shown in SEQ ID NO. 403; and the downstream primer with the sequence shown in SEQ ID NO. 404; The primer pairs for detecting RET gene DNA mutations include: the upstream primer and the downstream primer shown in SEQ ID NO. 357; the upstream primer and the downstream primer shown in SEQ ID NO. 358; the upstream primer and the downstream primer shown in SEQ ID NO. 359; the upstream primer and the downstream primer shown in SEQ ID NO. 360; the upstream primer and the downstream primer shown in SEQ ID NO. 361; the upstream primer and the downstream primer shown in SEQ ID NO. 362; the upstream primer and the downstream primer shown in SEQ ID NO. 363; the upstream primer and the downstream primer shown in SEQ ID NO. 364; the upstream primer and the downstream primer shown in SEQ ID NO. 365; the upstream primer and the downstream primer shown in SEQ ID NO. 366; the upstream primer and the downstream primer shown in SEQ ID NO. 367; the upstream primer and the downstream primer shown in SEQ ID NO. 368; the upstream primer and the downstream primer shown in SEQ ID NO. 369; and the upstream primer and the downstream primer shown in SEQ ID NO.

370. The downstream primer shown in SEQ ID NO. 372; the upstream primer shown in SEQ ID NO. 373 and the downstream primer shown in SEQ ID NO. 374; the upstream primer shown in SEQ ID NO. 375 and the downstream primer shown in SEQ ID NO. 376; the upstream primer shown in SEQ ID NO. 377 and the downstream primer shown in SEQ ID NO. 378; the upstream primer shown in SEQ ID NO. 379 and the downstream primer shown in SEQ ID NO. 380; the upstream primer shown in SEQ ID NO. 381 and the downstream primer shown in SEQ ID NO. 382; the upstream primer shown in SEQ ID NO. 383 and the downstream primer shown in SEQ ID NO. 384; The primer pairs for detecting CDKN2A gene DNA mutations include: the upstream primer shown in SEQ ID NO. 11 and the downstream primer shown in SEQ ID NO. 12; the upstream primer shown in SEQ ID NO. 13 and the downstream primer shown in SEQ ID NO. 14; the upstream primer shown in SEQ ID NO. 15 and the downstream primer shown in SEQ ID NO. 16; the upstream primer shown in SEQ ID NO. 17 and the downstream primer shown in SEQ ID NO. 18; the upstream primer shown in SEQ ID NO. 19 and the downstream primer shown in SEQ ID NO. 20; the upstream primer shown in SEQ ID NO. 21 and the downstream primer shown in SEQ ID NO. 22; the upstream primer shown in SEQ ID NO. 23 and the downstream primer shown in SEQ ID NO. 24; the upstream primer shown in SEQ ID NO. 25 and the downstream primer shown in SEQ ID NO. 26; the primer pairs shown in SEQ ID NO. 11 and SEQ ID NO. 12; the upstream primer shown in SEQ ID NO. 13 and the downstream primer shown in SEQ ID NO. 14; the upstream primer shown in SEQ ID NO. 15 and the downstream primer shown in SEQ ID NO. 16; the primer pairs shown in SEQ ID NO. 17 and the downstream primer shown in SEQ ID NO. 18; the upstream primer shown in SEQ ID NO. 19 and the downstream primer shown in SEQ ID NO. 20; the upstream primer shown in SEQ ID NO. 21 and the downstream primer shown in SEQ ID NO. 22; the upstream primer shown in SEQ ID NO. 23 and the downstream primer shown in SEQ ID NO. 24; the upstream primer shown in SEQ ID NO. 25 and the downstream primer shown in SEQ ID NO. 26; the primer pairs shown in SEQ ID NO. 17 and SEQ ID NO. 18; the upstream primer shown in SEQ ID NO. 19 and the downstream primer shown in SEQ ID NO. 20; the The upstream primer shown in NO. 27 and the downstream primer with the sequence shown in SEQ ID NO. 28; the upstream primer with the sequence shown in SEQ ID NO. 29 and the downstream primer with the sequence shown in SEQ ID NO. 30; the upstream primer with the sequence shown in SEQ ID NO. 31 and the downstream primer with the sequence shown in SEQ ID NO. 32; The primer pairs for detecting DICER1 gene DNA mutations include: an upstream primer with the sequence shown in SEQ ID NO. 41 and a downstream primer with the sequence shown in SEQ ID NO. 42; an upstream primer with the sequence shown in SEQ ID NO. 43 and a downstream primer with the sequence shown in SEQ ID NO. 44; and an upstream primer with the sequence shown in SEQ ID NO. 45 and a downstream primer with the sequence shown in SEQ ID NO.

46. The primer pairs for detecting EIF1AX gene DNA mutations include: the upstream primer and the downstream primer as shown in SEQ ID NO. 47; the upstream primer and the downstream primer as shown in SEQ ID NO. 48; the upstream primer and the downstream primer as shown in SEQ ID NO. 49; the upstream primer and the downstream primer as shown in SEQ ID NO. 50; the upstream primer and the downstream primer as shown in SEQ ID NO. 51; the upstream primer and the downstream primer as shown in SEQ ID NO. 52; the upstream primer and the downstream primer as shown in SEQ ID NO. 53; the upstream primer and the downstream primer as shown in SEQ ID NO. 54; the upstream primer and the downstream primer as shown in SEQ ID NO. 55; the upstream primer and the downstream primer as shown in SEQ ID NO. 56; the upstream primer and the downstream primer as shown in SEQ ID NO. 57; the upstream primer and the downstream primer as shown in SEQ ID NO. 58; the upstream primer and the downstream primer as shown in SEQ ID NO. 59; the upstream primer and the downstream primer as shown in SEQ ID NO. 60; the upstream primer and the downstream primer as shown in SEQ ID NO. 61; the primer as shown in SEQ ID NO. 62; the primer as shown in SEQ ID NO. 69; the primer as shown in SEQ ID NO. 60; the ... The upstream primer shown in SEQ ID NO. 63 and the downstream primer with the sequence shown in SEQ ID NO. 64; the upstream primer with the sequence shown in SEQ ID NO. 65 and the downstream primer with the sequence shown in SEQ ID NO. 66; the upstream primer with the sequence shown in SEQ ID NO. 67 and the downstream primer with the sequence shown in SEQ ID NO. 68; the upstream primer with the sequence shown in SEQ ID NO. 69 and the downstream primer with the sequence shown in SEQ ID NO. 70; the upstream primer with the sequence shown in SEQ ID NO. 71 and the downstream primer with the sequence shown in SEQ ID NO. 72; the upstream primer with the sequence shown in SEQ ID NO. 73 and the downstream primer with the sequence shown in SEQ ID NO. 74; the upstream primer with the sequence shown in SEQ ID NO. 75 and the downstream primer with the sequence shown in SEQ ID NO. 76; the upstream primer with the sequence shown in SEQ ID NO. 77 and the downstream primer with the sequence shown in SEQ ID NO. 78; the upstream primer with the sequence shown in SEQ ID NO. 79 and the downstream primer with the sequence shown in SEQ ID NO.

64. The downstream primer shown in SEQ ID NO. 80; the upstream primer shown in SEQ ID NO. 81 and the downstream primer shown in SEQ ID NO. 82; the upstream primer shown in SEQ ID NO. 83 and the downstream primer shown in SEQ ID NO. 84; the upstream primer shown in SEQ ID NO. 85 and the downstream primer shown in SEQ ID NO. 86; the sequence shown in SEQ ID NO.The upstream primer shown in SEQ ID NO. 87 and the downstream primer with the sequence shown in SEQ ID NO. 88; the upstream primer with the sequence shown in SEQ ID NO. 89 and the downstream primer with the sequence shown in SEQ ID NO. 90; the upstream primer with the sequence shown in SEQ ID NO. 91 and the downstream primer with the sequence shown in SEQ ID NO. 92; the upstream primer with the sequence shown in SEQ ID NO. 93 and the downstream primer with the sequence shown in SEQ ID NO. 94; the upstream primer with the sequence shown in SEQ ID NO. 95 and the downstream primer with the sequence shown in SEQ ID NO. 96; the upstream primer with the sequence shown in SEQ ID NO. 97 and the downstream primer with the sequence shown in SEQ ID NO. 98; the upstream primer with the sequence shown in SEQ ID NO. 99 and the downstream primer with the sequence shown in SEQ ID NO. 100; the upstream primer with the sequence shown in SEQ ID NO. 101 and the downstream primer with the sequence shown in SEQ ID NO. 102; the upstream primer with the sequence shown in SEQ ID NO. 103 and the downstream primer with the sequence shown in SEQ ID NO.

102. The downstream primer shown in SEQ ID NO. 104; the upstream primer with the sequence shown in SEQ ID NO. 105 and the downstream primer with the sequence shown in SEQ ID NO. 106; the upstream primer with the sequence shown in SEQ ID NO. 107 and the downstream primer with the sequence shown in SEQ ID NO. 108; The primer pairs for detecting GNAS gene DNA mutations include: an upstream primer with the sequence shown in SEQ ID NO. 109 and a downstream primer with the sequence shown in SEQ ID NO. 110; an upstream primer with the sequence shown in SEQ ID NO. 111 and a downstream primer with the sequence shown in SEQ ID NO. 112; The primer pairs for detecting HRAS gene DNA mutations include: an upstream primer with the sequence shown in SEQ ID NO. 113 and a downstream primer with the sequence shown in SEQ ID NO. 114; an upstream primer with the sequence shown in SEQ ID NO. 115 and a downstream primer with the sequence shown in SEQ ID NO. 116; an upstream primer with the sequence shown in SEQ ID NO. 117 and a downstream primer with the sequence shown in SEQ ID NO. 118; and an upstream primer with the sequence shown in SEQ ID NO. 119 and a downstream primer with the sequence shown in SEQ ID NO.

120. The primer pairs for detecting PIK3CA gene DNA mutations include: the upstream primer and the downstream primer shown in SEQ ID NO. 137; the upstream primer and the downstream primer shown in SEQ ID NO. 138; the upstream primer and the downstream primer shown in SEQ ID NO. 139; the upstream primer and the downstream primer shown in SEQ ID NO. 140; the upstream primer and the downstream primer shown in SEQ ID NO. 141; the upstream primer and the downstream primer shown in SEQ ID NO. 142; the upstream primer and the downstream primer shown in SEQ ID NO. 143; the upstream primer and the downstream primer shown in SEQ ID NO. 144; the upstream primer and the downstream primer shown in SEQ ID NO. 145; the upstream primer and the downstream primer shown in SEQ ID NO. 146; the upstream primer and the downstream primer shown in SEQ ID NO. 147; the upstream primer and the downstream primer shown in SEQ ID NO. 148; the upstream primer and the downstream primer shown in SEQ ID NO. 149; and the upstream primer and the downstream primer shown in SEQ ID NO. 150; The downstream primer shown in SEQ ID NO. 152; the upstream primer shown in SEQ ID NO. 153 and the downstream primer shown in SEQ ID NO. 154; the upstream primer shown in SEQ ID NO. 155 and the downstream primer shown in SEQ ID NO. 156; the upstream primer shown in SEQ ID NO. 157 and the downstream primer shown in SEQ ID NO. 158; the upstream primer shown in SEQ ID NO. 159 and the downstream primer shown in SEQ ID NO. 160; the upstream primer shown in SEQ ID NO. 161 and the downstream primer shown in SEQ ID NO. 162; the upstream primer shown in SEQ ID NO. 163 and the downstream primer shown in SEQ ID NO. 164; the upstream primer shown in SEQ ID NO. 165 and the downstream primer shown in SEQ ID NO. 166; the upstream primer shown in SEQ ID NO. 167 and the downstream primer shown in SEQ ID NO.

158. The downstream primer shown in SEQ ID NO. 168; the upstream primer shown in SEQ ID NO. 169 and the downstream primer shown in SEQ ID NO. 170; the upstream primer shown in SEQ ID NO. 171 and the downstream primer shown in SEQ ID NO. 172; the upstream primer shown in SEQ ID NO. 173 and the downstream primer shown in SEQ ID NO. 174; and the sequence shown in SEQ ID NO.The upstream primer shown in SEQ ID NO. 175 and the downstream primer with the sequence shown in SEQ ID NO. 176; the upstream primer with the sequence shown in SEQ ID NO. 177 and the downstream primer with the sequence shown in SEQ ID NO. 178; the upstream primer with the sequence shown in SEQ ID NO. 179 and the downstream primer with the sequence shown in SEQ ID NO. 180; the upstream primer with the sequence shown in SEQ ID NO. 181 and the downstream primer with the sequence shown in SEQ ID NO. 182; the upstream primer with the sequence shown in SEQ ID NO. 183 and the downstream primer with the sequence shown in SEQ ID NO. 184; the upstream primer with the sequence shown in SEQ ID NO. 185 and the downstream primer with the sequence shown in SEQ ID NO. 186; the upstream primer with the sequence shown in SEQ ID NO. 187 and the downstream primer with the sequence shown in SEQ ID NO. 188; the upstream primer with the sequence shown in SEQ ID NO. 189 and the downstream primer with the sequence shown in SEQ ID NO. 190; the sequence shown in SEQ ID NO. The upstream primer shown in SEQ ID NO. 191 and the downstream primer with the sequence shown in SEQ ID NO. 192; the upstream primer with the sequence shown in SEQ ID NO. 193 and the downstream primer with the sequence shown in SEQ ID NO. 194; the upstream primer with the sequence shown in SEQ ID NO. 195 and the downstream primer with the sequence shown in SEQ ID NO. 196; the upstream primer with the sequence shown in SEQ ID NO. 197 and the downstream primer with the sequence shown in SEQ ID NO. 198; the upstream primer with the sequence shown in SEQ ID NO. 199 and the downstream primer with the sequence shown in SEQ ID NO. 200; the upstream primer with the sequence shown in SEQ ID NO. 201 and the downstream primer with the sequence shown in SEQ ID NO. 202; the upstream primer with the sequence shown in SEQ ID NO. 203 and the downstream primer with the sequence shown in SEQ ID NO. 204; the upstream primer with the sequence shown in SEQ ID NO. 205 and the downstream primer with the sequence shown in SEQ ID NO. 206; and the sequence shown in SEQ ID NO. The upstream primer shown in SEQ ID NO. 207 and the downstream primer with the sequence shown in SEQ ID NO. 208; the upstream primer with the sequence shown in SEQ ID NO. 209 and the downstream primer with the sequence shown in SEQ ID NO. 210; the upstream primer with the sequence shown in SEQ ID NO. 211 and the downstream primer with the sequence shown in SEQ ID NO. 212; the upstream primer with the sequence shown in SEQ ID NO. 213 and the downstream primer with the sequence shown in SEQ ID NO. 214; and the sequence shown in SEQ ID NO.The upstream primer shown in SEQ ID NO. 215 and the downstream primer with the sequence shown in SEQ ID NO. 216; the upstream primer with the sequence shown in SEQ ID NO. 217 and the downstream primer with the sequence shown in SEQ ID NO. 218; the upstream primer with the sequence shown in SEQ ID NO. 219 and the downstream primer with the sequence shown in SEQ ID NO. 220; the upstream primer with the sequence shown in SEQ ID NO. 221 and the downstream primer with the sequence shown in SEQ ID NO. 222; the upstream primer with the sequence shown in SEQ ID NO. 223 and the downstream primer with the sequence shown in SEQ ID NO. 224; the upstream primer with the sequence shown in SEQ ID NO. 225 and the downstream primer with the sequence shown in SEQ ID NO. 226; the upstream primer with the sequence shown in SEQ ID NO. 227 and the downstream primer with the sequence shown in SEQ ID NO. 228; the upstream primer with the sequence shown in SEQ ID NO. 229 and the downstream primer with the sequence shown in SEQ ID NO. 230; and the sequence shown in SEQ ID NO. The upstream primer shown in SEQ ID NO. 231 and the downstream primer with the sequence shown in SEQ ID NO. 232; the upstream primer with the sequence shown in SEQ ID NO. 233 and the downstream primer with the sequence shown in SEQ ID NO. 234; the upstream primer with the sequence shown in SEQ ID NO. 235 and the downstream primer with the sequence shown in SEQ ID NO.

236. The primer pairs for detecting PTEN gene DNA mutations include: the upstream primer and the downstream primer shown in SEQ ID NO. 237; the upstream primer and the downstream primer shown in SEQ ID NO. 238; the upstream primer and the downstream primer shown in SEQ ID NO. 239; the upstream primer and the downstream primer shown in SEQ ID NO. 240; the upstream primer and the downstream primer shown in SEQ ID NO. 241; the upstream primer and the downstream primer shown in SEQ ID NO. 242; the upstream primer and the downstream primer shown in SEQ ID NO. 243; the upstream primer and the downstream primer shown in SEQ ID NO. 244; the upstream primer and the downstream primer shown in SEQ ID NO. 245; the upstream primer and the downstream primer shown in SEQ ID NO. 246; the upstream primer and the downstream primer shown in SEQ ID NO. 247; the upstream primer and the downstream primer shown in SEQ ID NO. 248; the upstream primer and the downstream primer shown in SEQ ID NO. 249; and the upstream primer and the downstream primer shown in SEQ ID NO.

250. The downstream primer shown in SEQ ID NO. 252; the upstream primer shown in SEQ ID NO. 253 and the downstream primer shown in SEQ ID NO. 254; the upstream primer shown in SEQ ID NO. 255 and the downstream primer shown in SEQ ID NO. 256; the upstream primer shown in SEQ ID NO. 257 and the downstream primer shown in SEQ ID NO. 258; the upstream primer shown in SEQ ID NO. 259 and the downstream primer shown in SEQ ID NO. 260; the upstream primer shown in SEQ ID NO. 261 and the downstream primer shown in SEQ ID NO. 262; the upstream primer shown in SEQ ID NO. 263 and the downstream primer shown in SEQ ID NO. 264; the upstream primer shown in SEQ ID NO. 265 and the downstream primer shown in SEQ ID NO. 266; the upstream primer shown in SEQ ID NO. 267 and the downstream primer shown in SEQ ID NO.

254. The downstream primer shown in SEQ ID NO. 268; the upstream primer shown in SEQ ID NO. 269 and the downstream primer shown in SEQ ID NO. 270; the upstream primer shown in SEQ ID NO. 271 and the downstream primer shown in SEQ ID NO. 272; the upstream primer shown in SEQ ID NO. 273 and the downstream primer shown in SEQ ID NO. 274; and the sequence shown in SEQ ID NO.The upstream primer shown in SEQ ID NO. 275 and the downstream primer shown in SEQ ID NO. 276; the upstream primer shown in SEQ ID NO. 277 and the downstream primer shown in SEQ ID NO. 278; the upstream primer shown in SEQ ID NO. 279 and the downstream primer shown in SEQ ID NO. 280; the upstream primer shown in SEQ ID NO. 281 and the downstream primer shown in SEQ ID NO. 282; the upstream primer shown in SEQ ID NO. 283 and the downstream primer shown in SEQ ID NO. 284; the upstream primer shown in SEQ ID NO. 285 and the downstream primer shown in SEQ ID NO. 286; the upstream primer shown in SEQ ID NO. 287 and the downstream primer shown in SEQ ID NO. 288; the upstream primer shown in SEQ ID NO. 289 and the downstream primer shown in SEQ ID NO. 290; the sequence shown in SEQ ID NO. The upstream primer shown in SEQ ID NO. 291 and the downstream primer with the sequence shown in SEQ ID NO. 292; the upstream primer with the sequence shown in SEQ ID NO. 293 and the downstream primer with the sequence shown in SEQ ID NO. 294; the upstream primer with the sequence shown in SEQ ID NO. 295 and the downstream primer with the sequence shown in SEQ ID NO. 296; the upstream primer with the sequence shown in SEQ ID NO. 297 and the downstream primer with the sequence shown in SEQ ID NO. 298; the upstream primer with the sequence shown in SEQ ID NO. 299 and the downstream primer with the sequence shown in SEQ ID NO. 300; the upstream primer with the sequence shown in SEQ ID NO. 301 and the downstream primer with the sequence shown in SEQ ID NO. 302; the upstream primer with the sequence shown in SEQ ID NO. 303 and the downstream primer with the sequence shown in SEQ ID NO. 304; the upstream primer with the sequence shown in SEQ ID NO. 305 and the downstream primer with the sequence shown in SEQ ID NO. 306; and the sequence shown in SEQ ID NO. The upstream primer shown in SEQ ID NO. 307 and the downstream primer with the sequence shown in SEQ ID NO. 308; the upstream primer with the sequence shown in SEQ ID NO. 309 and the downstream primer with the sequence shown in SEQ ID NO. 310; the upstream primer with the sequence shown in SEQ ID NO. 311 and the downstream primer with the sequence shown in SEQ ID NO. 312; the upstream primer with the sequence shown in SEQ ID NO. 313 and the downstream primer with the sequence shown in SEQ ID NO. 314; and the sequence shown in SEQ ID NO.The upstream primer shown in SEQ ID NO. 315 and the downstream primer shown in SEQ ID NO. 316; the upstream primer shown in SEQ ID NO. 317 and the downstream primer shown in SEQ ID NO. 318; the upstream primer shown in SEQ ID NO. 319 and the downstream primer shown in SEQ ID NO. 320; the upstream primer shown in SEQ ID NO. 321 and the downstream primer shown in SEQ ID NO. 322; the upstream primer shown in SEQ ID NO. 323 and the downstream primer shown in SEQ ID NO. 324; the upstream primer shown in SEQ ID NO. 325 and the downstream primer shown in SEQ ID NO. 326; the upstream primer shown in SEQ ID NO. 327 and the downstream primer shown in SEQ ID NO. 328; the upstream primer shown in SEQ ID NO. 329 and the downstream primer shown in SEQ ID NO. 330; and the sequence shown in SEQ ID NO. The upstream primer shown in SEQ ID NO. 331 and the downstream primer with the sequence shown in SEQ ID NO. 332; the upstream primer with the sequence shown in SEQ ID NO. 333 and the downstream primer with the sequence shown in SEQ ID NO. 334; the upstream primer with the sequence shown in SEQ ID NO. 335 and the downstream primer with the sequence shown in SEQ ID NO. 336; the upstream primer with the sequence shown in SEQ ID NO. 337 and the downstream primer with the sequence shown in SEQ ID NO. 338; the upstream primer with the sequence shown in SEQ ID NO. 339 and the downstream primer with the sequence shown in SEQ ID NO. 340; the upstream primer with the sequence shown in SEQ ID NO. 341 and the downstream primer with the sequence shown in SEQ ID NO. 342; the upstream primer with the sequence shown in SEQ ID NO. 343 and the downstream primer with the sequence shown in SEQ ID NO. 344; the upstream primer with the sequence shown in SEQ ID NO. 345 and the downstream primer with the sequence shown in SEQ ID NO. 346; the sequence shown in SEQ ID NO. The upstream primer shown in SEQ ID NO. 347 and the downstream primer with the sequence shown in SEQ ID NO. 348; the upstream primer with the sequence shown in SEQ ID NO. 349 and the downstream primer with the sequence shown in SEQ ID NO. 350; the upstream primer with the sequence shown in SEQ ID NO. 351 and the downstream primer with the sequence shown in SEQ ID NO. 352; the upstream primer with the sequence shown in SEQ ID NO. 353 and the downstream primer with the sequence shown in SEQ ID NO.

354. The primer pairs for detecting TSHR gene DNA mutations include: the upstream primer and the downstream primer shown in SEQ ID NO. 405; the upstream primer and the downstream primer shown in SEQ ID NO. 406; the upstream primer and the downstream primer shown in SEQ ID NO. 407; the upstream primer and the downstream primer shown in SEQ ID NO. 408; the upstream primer and the downstream primer shown in SEQ ID NO. 409; the upstream primer and the downstream primer shown in SEQ ID NO. 410; the upstream primer and the downstream primer shown in SEQ ID NO. 411; the upstream primer and the downstream primer shown in SEQ ID NO. 412; the upstream primer and the downstream primer shown in SEQ ID NO. 413; the upstream primer and the downstream primer shown in SEQ ID NO. 414; the upstream primer and the downstream primer shown in SEQ ID NO. 415; the upstream primer and the downstream primer shown in SEQ ID NO. 416; the upstream primer and the downstream primer shown in SEQ ID NO. 417; and the upstream primer and the downstream primer shown in SEQ ID NO. 418; and the upstream primer and the downstream primer shown in SEQ ID NO.

419. The downstream primer shown in SEQ ID NO. 420; the upstream primer shown in SEQ ID NO. 421 and the downstream primer shown in SEQ ID NO. 422; the upstream primer shown in SEQ ID NO. 423 and the downstream primer shown in SEQ ID NO. 424; the upstream primer shown in SEQ ID NO. 425 and the downstream primer shown in SEQ ID NO. 426; the upstream primer shown in SEQ ID NO. 427 and the downstream primer shown in SEQ ID NO. 428; the upstream primer shown in SEQ ID NO. 429 and the downstream primer shown in SEQ ID NO. 430; the upstream primer shown in SEQ ID NO. 431 and the downstream primer shown in SEQ ID NO. 432; the upstream primer shown in SEQ ID NO. 433 and the downstream primer shown in SEQ ID NO. 434; the upstream primer shown in SEQ ID NO. 435 and the downstream primer shown in SEQ ID NO.

426. The downstream primer shown in SEQ ID NO. 436; the upstream primer shown in SEQ ID NO. 437 and the downstream primer shown in SEQ ID NO. 438; the upstream primer shown in SEQ ID NO. 439 and the downstream primer shown in SEQ ID NO. 440; the upstream primer shown in SEQ ID NO. 441 and the downstream primer shown in SEQ ID NO. 442; and the sequence shown in SEQ ID NO.The upstream primer shown in SEQ ID NO. 443 and the downstream primer with the sequence shown in SEQ ID NO. 444; the upstream primer with the sequence shown in SEQ ID NO. 445 and the downstream primer with the sequence shown in SEQ ID NO. 446; the upstream primer with the sequence shown in SEQ ID NO. 447 and the downstream primer with the sequence shown in SEQ ID NO. 448; the upstream primer with the sequence shown in SEQ ID NO. 449 and the downstream primer with the sequence shown in SEQ ID NO. 450; the upstream primer with the sequence shown in SEQ ID NO. 451 and the downstream primer with the sequence shown in SEQ ID NO. 452; the upstream primer with the sequence shown in SEQ ID NO. 453 and the downstream primer with the sequence shown in SEQ ID NO. 454; the upstream primer with the sequence shown in SEQ ID NO. 455 and the downstream primer with the sequence shown in SEQ ID NO. 456; the upstream primer with the sequence shown in SEQ ID NO. 457 and the downstream primer with the sequence shown in SEQ ID NO. 458; and the sequence shown in SEQ ID NO. The upstream primer shown in SEQ ID NO. 459 and the downstream primer with the sequence shown in SEQ ID NO. 460; the upstream primer with the sequence shown in SEQ ID NO. 461 and the downstream primer with the sequence shown in SEQ ID NO. 462; the upstream primer with the sequence shown in SEQ ID NO. 463 and the downstream primer with the sequence shown in SEQ ID NO. 464; the upstream primer with the sequence shown in SEQ ID NO. 465 and the downstream primer with the sequence shown in SEQ ID NO. 466; the upstream primer with the sequence shown in SEQ ID NO. 467 and the downstream primer with the sequence shown in SEQ ID NO. 468; the upstream primer with the sequence shown in SEQ ID NO. 469 and the downstream primer with the sequence shown in SEQ ID NO. 470; the upstream primer with the sequence shown in SEQ ID NO. 471 and the downstream primer with the sequence shown in SEQ ID NO.

472. The primer pair for detecting ARAF gene DNA mutations includes: an upstream primer with the sequence shown in SEQ ID NO. 3 and a downstream primer with the sequence shown in SEQ ID NO. 4; The primer pair for detecting AKT1 gene DNA mutations includes: an upstream primer with the sequence shown in SEQ ID NO. 1 and a downstream primer with the sequence shown in SEQ ID NO. 2; The primer pairs for detecting CTNNB1 gene DNA mutations include: an upstream primer with the sequence shown in SEQ ID NO. 33 and a downstream primer with the sequence shown in SEQ ID NO. 34; an upstream primer with the sequence shown in SEQ ID NO. 35 and a downstream primer with the sequence shown in SEQ ID NO. 36; an upstream primer with the sequence shown in SEQ ID NO. 37 and a downstream primer with the sequence shown in SEQ ID NO. 38; and an upstream primer with the sequence shown in SEQ ID NO. 39 and a downstream primer with the sequence shown in SEQ ID NO.

40. The primer pair for detecting RAF1 gene DNA mutations includes: an upstream primer with the sequence shown in SEQ ID NO. 355 and a downstream primer with the sequence shown in SEQ ID NO. 356; The ALK gene RNA fusion variant includes RNA fusion variants of the ALK gene and the EML4, KIF5B or TPM3 genes, and the primer pair includes: an upstream primer with sequences as shown in SEQ ID NO. 551~556, SEQ ID NO. 561~566, SEQ ID NO. 581~584 and a downstream primer with sequences as shown in SEQ ID NO. 473~480; The BRAF gene RNA fusion mutation is an RNA fusion mutation of the BRAF gene and the AKAP9 gene, and the primer pair includes: an upstream primer with the sequence shown in SEQ ID NO. 485~490 and a downstream primer with the sequence shown in SEQ ID NO. 481~484 and SEQ ID NO. 589~590; The NTRK1 gene RNA fusion variant is an RNA fusion variant of the NTRK1 gene and the TPM3 gene, and the primer pair includes: an upstream primer with a sequence as shown in SEQ ID NO. 581~584 and a downstream primer with a sequence as shown in SEQ ID NO. 491~508; The NTRK2 gene RNA fusion variant is an RNA fusion variant of the NTRK2 gene and the BCR gene, and the primer pair includes: an upstream primer with a sequence as shown in SEQ ID NO. 591~592 and a downstream primer with a sequence as shown in SEQ ID NO. 509~518; The NTRK3 gene RNA fusion variant is an RNA fusion variant of the NTRK3 gene and the ETV6 gene. The primer pair includes: an upstream primer with sequences as shown in SEQ ID NO. 523~530 and SEQ ID NO. 557~560 and a downstream primer with sequences as shown in SEQ ID NO. 519~522 and SEQ ID NO. 531~532. The PPARG gene RNA fusion variant is an RNA fusion variant of the PPARG gene and the PAX8 gene, and the primer pair includes: an upstream primer with sequences shown in SEQ ID NO. 573~578 and a downstream primer with sequences shown in SEQ ID NO. 587~588; The RET gene RNA fusion variant includes RNA fusion variants of the RET gene and the KIF5B, CCDC6, NCOA4, or PRKAR1A genes, and the primer pair includes: an upstream primer with sequences as shown in SEQ ID NO. 561~566, SEQ ID NO. 545~550, SEQ ID NO. 567~572, and SEQ ID NO. 579~580, and a downstream primer with sequences as shown in SEQ ID NO. 533~538; The THADA gene RNA fusion mutation is an RNA fusion mutation of the THADA gene and the IGF2BP3 gene, and the primer pair includes: an upstream primer with a sequence as shown in SEQ ID NO. 539~544 and a downstream primer with a sequence as shown in SEQ ID NO. 585~586; In the primer composition, at least 50% of the upstream primers have a nucleotide fragment with the sequence shown in SEQ ID NO. 593 attached to their 5' ends, and the corresponding downstream primers have a nucleotide fragment with the sequence shown in SEQ ID NO. 594 attached to their 3' ends.

2. The use of the primer composition as described in claim 1 in the preparation of a product for detecting gene mutations related to benign or malignant thyroid nodules.

3. The application as described in claim 2, characterized in that, The product in question is a reagent kit.

4. A kit for detecting gene mutations related to benign and malignant thyroid nodules, characterized in that, The kit includes the primer composition as described in claim 1.

5. A system for detecting gene mutations related to benign and malignant thyroid nodules, characterized in that, include: Detection module: The detection module includes a multiplex amplification module for genes related to benign and malignant thyroid nodules, a library construction module, and a sequencing module; the multiplex amplification module for genes related to benign and malignant thyroid nodules includes the primer composition as described in claim 1 or the kit as described in claim 4; Data analysis module: The data analysis module analyzes the sequencing results of the sequencing module, including filtering data below the quality control threshold, removing adapters, aligning sequences to the corresponding positions in the reference genome data, annotating gene mutations, and performing statistical analysis of the corresponding data.

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