Primer composition, kit and method for detecting benign and malignant thyroid nodule related gene mutation

Abstract

The invention relates to a primer composition, kit and method for detecting benign and malignant thyroid nodule related gene mutation, and the primer composition comprises a primer pair for detecting benign and malignant thyroid nodule related gene DNA mutation and RNA fusion variation. The primer pair can be independently amplified, and can be used for performing efficient, high-sensitivity and high-specificity multiple amplification on DNA and cDNA templates to be detected under the same PCR reaction system and reaction program, and then the gene mutation condition of a related region can be obtained by constructing a library and sequencing, so as to assist clinical diagnosis of benign and malignant thyroid nodules. Moreover, specific nucleotide sequences are respectively connected to the 5'end of the upstream primer and the 3 'end of the corresponding downstream primer in a proper proportion in the primer composition, so that the homogeneity and specificity of multiple PCR amplification can be further improved, and the detected nucleic acid input amount and the lowest detection limit of mutation frequency (as low as 0.5%) are effectively reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology and relates to gene mutation detection, and specifically to a primer composition, a kit and a method for detecting gene mutations associated with benign and malignant thyroid nodules. Background Art

[0002] Currently, ultrasound-guided fine-needle aspiration cytology (US-FNAB) is used as the gold standard for preoperative diagnosis of thyroid tumors in clinical practice, but 10%-40% of samples cannot be clearly distinguished as benign or malignant by US-FNAB. For thyroid nodules that cannot be clearly distinguished as benign or malignant, it is necessary to add molecular testing of hotspot genes of thyroid tumors on top of US-FNAB examination.

[0003] Conventional tumor molecular detection generally uses hybrid capture library construction, of which two forms of detection are the most common: the first is NGS large panel detection, which can cover hundreds of genes / sites in one test, but the sequencing cost is high due to the large detection area, the detection price is expensive, and the detection of genes related to thyroid malignant tumors is not focused enough, resulting in data waste. The second method is a small panel test customized for thyroid tumors, which can detect 10-15 genes related to thyroid tumors (including fixed hot spot fusion gene sites). The number of genes detected is relatively small, and the starting amount of nucleic acid required for hybrid capture library construction is high (at least 50-100ng). Most thyroid fine needle puncture samples are small in size and low in nucleic acid concentration, making it difficult to meet the detection requirements. In addition, the hybrid capture library construction method is complex to operate, the experimental process is long, and the entire detection cycle is 7-8 days; small panels are prone to off-target during the capture and elution process, and the detection sensitivity is limited, so it is not clinically universal.

[0004] In addition to the hybridization capture library construction method, amplicon library construction is currently available for tumor molecular detection. Compared with the hybridization capture library construction method, the initial nucleic acid input of this method is lower, but the PCR reaction uniformity of the amplicon library construction is disadvantageous. Conventional multiple amplicon reactions have amplification preferences and are prone to preferentially amplify short fragments, resulting in nonspecific amplification. A large number of nonspecific fragments in the same system will consume the enzymes and substrates of the PCR reaction, resulting in a decrease in the amplification efficiency of the target fragment and poor overall amplification uniformity. Currently, there is no technical route for molecular detection of thyroid fine needle puncture samples through the above library construction method.

[0005] Therefore, the market needs a molecular detection method with good uniformity, high sensitivity, low nucleic acid input requirements and price advantages to help clinically differentiate between benign and malignant thyroid nodules, reduce the surgical resection rate of thyroid nodules, and help with precise diagnosis and treatment while reducing the financial pressure on patients. Summary of the invention

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

[0007] The first aspect of the present invention is to provide a primer composition for detecting gene mutations associated with benign and malignant thyroid nodules, including primer pairs for detecting BRAF and KRAS gene DNA mutations;

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

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

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

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

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

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

[0014] The primer pair for detecting RET gene DNA mutation is selected from at least one of the following pairs: an upstream primer with a sequence as shown in SEQ ID NO.357 and a downstream primer with a sequence as shown in SEQ ID NO.358; an upstream primer with a sequence as shown in SEQ ID NO.359 and a downstream primer with a sequence as shown in SEQ ID NO.360; an upstream primer with a sequence as shown in SEQ ID NO.361 and a downstream primer with a sequence as shown in SEQ ID NO.362; an upstream primer with a sequence as shown in SEQ ID NO.363 and a downstream primer with a sequence as shown in SEQ ID NO.364; an upstream primer with a sequence as shown in SEQ ID NO.365 and a downstream primer with a sequence as shown in SEQ ID NO.366; an upstream primer with a sequence as shown in SEQ ID NO.367 and a downstream primer with a sequence as shown in SEQ ID NO.368; an upstream primer with a sequence as shown in SEQ ID NO.369 and a downstream primer with a sequence as shown in SEQ ID NO.370; an upstream primer with a sequence as shown in SEQ ID NO.371 and a downstream primer with a sequence as shown in SEQ ID NO.372; The upstream primer shown in 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 comprises at least one of primer pairs for detecting DNA mutations in CDKN2A, DICER1, EIF1AX, GNAS, HRAS, PIK3CA, PTEN, TSHR, ARAF, AKT1, CTNNB1, and RAF1 genes;

[0016] The primer pair for detecting the DNA mutation of the CDKN2A gene is selected from at least one of the following pairs: an upstream primer having a sequence as shown in SEQ ID NO.11 and a downstream primer having a sequence as shown in SEQ ID NO.12; an upstream primer having a sequence as shown in SEQ ID NO.13 and a downstream primer having a sequence as shown in SEQ ID NO.14; an upstream primer having a sequence as shown in SEQ ID NO.15 and a downstream primer having a sequence as shown in SEQ ID NO.16; an upstream primer having a sequence as shown in SEQ ID NO.17 and a downstream primer having a sequence as shown in SEQ ID NO.18; an upstream primer having a sequence as shown in SEQ ID NO.19 and a downstream primer having a sequence as shown in SEQ ID NO.20; an upstream primer having a sequence as shown in SEQ ID NO.21 and a downstream primer having a sequence as shown in SEQ ID NO.22; an upstream primer having a sequence as shown in SEQ ID NO.23 and a downstream primer having a sequence as shown in SEQ ID NO.24; an upstream primer having a sequence as shown in SEQ ID NO.25 and a downstream primer having a sequence as shown in SEQ ID NO.26; an upstream primer having a sequence as shown in SEQ ID NO.27 and a downstream primer having a sequence as shown in SEQ ID NO. A downstream primer as shown in SEQ ID NO.28; an upstream primer as shown in SEQ ID NO.29 and a downstream primer as shown in SEQ ID NO.30; an upstream primer as shown in SEQ ID NO.31 and a downstream primer as shown in SEQ ID NO.32;

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

[0018] The primer pair for detecting the EIF1AX gene DNA mutation is selected from at least one of the following pairs: an upstream primer having a sequence as shown in SEQ ID NO.47 and a downstream primer having a sequence as shown in SEQ ID NO.48; an upstream primer having a sequence as shown in SEQ ID NO.49 and a downstream primer having a sequence as shown in SEQ ID NO.50; an upstream primer having a sequence as shown in SEQ ID NO.51 and a downstream primer having a sequence as shown in SEQ ID NO.52; an upstream primer having a sequence as shown in SEQ ID NO.53 and a downstream primer having a sequence as shown in SEQ ID NO.54; an upstream primer having a sequence as shown in SEQ ID NO.55 and a downstream primer having a sequence as shown in SEQ ID NO.56; an upstream primer having a sequence as shown in SEQ ID NO.57 and a downstream primer having a sequence as shown in SEQ ID NO.58; an upstream primer having a sequence as shown in SEQ ID NO.59 and a downstream primer having a sequence as shown in SEQ ID NO.60; an upstream primer having a sequence as shown in SEQ ID NO.61 and a downstream primer having a sequence as shown in SEQ ID NO.62; an upstream primer having a sequence as shown in SEQ ID NO.63 and a downstream primer having a sequence as shown in SEQ ID NO. NO.64; an upstream primer having a sequence as shown in SEQ ID NO.65 and a downstream primer having a sequence as shown in SEQ ID NO.66; an upstream primer having a sequence as shown in SEQ ID NO.67 and a downstream primer having a sequence as shown in SEQ ID NO.68; an upstream primer having a sequence as shown in SEQ ID NO.69 and a downstream primer having a sequence as shown in SEQ ID NO.70; an upstream primer having a sequence as shown in SEQ ID NO.71 and a downstream primer having a sequence as shown in SEQ ID NO.72; an upstream primer having a sequence as shown in SEQ ID NO.73 and a downstream primer having a sequence as shown in SEQ ID NO.74; an upstream primer having a sequence as shown in SEQ ID NO.75 and a downstream primer having a sequence as shown in SEQ ID NO.76; an upstream primer having a sequence as shown in SEQ ID NO.77 and a downstream primer having a sequence as shown in SEQ ID NO.78; an upstream primer having a sequence as shown in SEQ ID NO.79 and a downstream primer having a sequence as shown in SEQ ID NO.80; an upstream primer having a sequence as shown in SEQ ID NO.81 and a downstream primer having a sequence as 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 upstream primer shown in SEQ ID NO.87 and the downstream primer shown in SEQ ID NO.88; an upstream primer having a sequence as shown in SEQ ID NO.89 and a downstream primer having a sequence as shown in SEQ ID NO.90; an upstream primer having a sequence as shown in SEQ ID NO.91 and a downstream primer having a sequence as shown in SEQ ID NO.92; an upstream primer having a sequence as shown in SEQ ID NO.93 and a downstream primer having a sequence as shown in SEQ ID NO.94; an upstream primer having a sequence as shown in SEQ ID NO.95 and a downstream primer having a sequence as shown in SEQ ID NO.96; an upstream primer having a sequence as shown in SEQ ID NO.97 and a downstream primer having a sequence as shown in SEQ ID NO.98; an upstream primer having a sequence as shown in SEQ ID NO.99 and a downstream primer having a sequence as shown in SEQ ID NO.100; an upstream primer having a sequence as shown in SEQ ID NO.101 and a downstream primer having a sequence as shown in SEQ ID NO.102; an upstream primer having a sequence as shown in SEQ ID NO.103 and a downstream primer having a sequence as shown in SEQ ID NO.104; an upstream primer having a sequence as shown in SEQ ID NO.105 and a downstream primer having a sequence as shown in SEQ ID NO. A downstream primer as shown in SEQ ID NO.106; an upstream primer as shown in SEQ ID NO.107 and a downstream primer as shown in SEQ ID NO.108;.

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

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

[0021] The primer pair for detecting the PIK3CA gene DNA mutation is selected from at least one of the following pairs: an upstream primer with a sequence as shown in SEQ ID NO.137 and a downstream primer with a sequence as shown in SEQ ID NO.138; an upstream primer with a sequence as shown in SEQ ID NO.139 and a downstream primer with a sequence as shown in SEQ ID NO.140; an upstream primer with a sequence as shown in SEQ ID NO.141 and a downstream primer with a sequence as shown in SEQ ID NO.142; an upstream primer with a sequence as shown in SEQ ID NO.143 and a downstream primer with a sequence as shown in SEQ ID NO.144; an upstream primer with a sequence as shown in SEQ ID NO.145 and a downstream primer with a sequence as shown in SEQ ID NO.146; an upstream primer with a sequence as shown in SEQ ID NO.147 and a downstream primer with a sequence as shown in SEQ ID NO.148; an upstream primer with a sequence as shown in SEQ ID NO.149 and a downstream primer with a sequence as shown in SEQ ID NO.150; an upstream primer with a sequence as shown in SEQ ID NO.151 and a downstream primer with a sequence as shown in SEQ ID NO.152; an upstream primer as shown in SEQ ID NO.153 and a downstream primer as shown in SEQ ID NO.154; an upstream primer as shown in SEQ ID NO.155 and a downstream primer as shown in SEQ ID NO.156; an upstream primer as shown in SEQ ID NO.157 and a downstream primer as shown in SEQ ID NO.158; an upstream primer as shown in SEQ ID NO.159 and a downstream primer as shown in SEQ ID NO.160; an upstream primer as shown in SEQ ID NO.161 and a downstream primer as shown in SEQ ID NO.162; an upstream primer as shown in SEQ ID NO.163 and a downstream primer as shown in SEQ ID NO.164; an upstream primer as shown in SEQ ID NO.165 and a downstream primer as shown in SEQ ID NO.166; an upstream primer as shown in SEQ ID NO.167 and a downstream primer as shown in SEQ ID NO.168; an upstream primer as shown in SEQ ID NO.169 and a downstream primer as shown in SEQ ID NO.170; NO.170; an upstream primer having a sequence as shown in SEQ ID NO.171 and a downstream primer having a sequence as shown in SEQ ID NO.172; an upstream primer having a sequence as shown in SEQ ID NO.173 and a downstream primer having a sequence as shown in SEQ ID NO.174; an upstream primer having a sequence as shown in SEQ ID NO.175 and a downstream primer having a sequence as shown in SEQ ID NO.176; a downstream primer having a sequence as shown in SEQ ID NO.an upstream primer as shown in SEQ ID NO.177 and a downstream primer as shown in SEQ ID NO.178; an upstream primer as shown in SEQ ID NO.179 and a downstream primer as shown in SEQ ID NO.180; an upstream primer as shown in SEQ ID NO.181 and a downstream primer as shown in SEQ ID NO.182; an upstream primer as shown in SEQ ID NO.183 and a downstream primer as shown in SEQ ID NO.184; an upstream primer as shown in SEQ ID NO.185 and a downstream primer as shown in SEQ ID NO.186; an upstream primer as shown in SEQ ID NO.187 and a downstream primer as shown in SEQ ID NO.188; an upstream primer as shown in SEQ ID NO.189 and a downstream primer as shown in SEQ ID NO.190; an upstream primer as shown in SEQ ID NO.191 and a downstream primer as shown in SEQ ID NO.192; an upstream primer as shown in SEQ ID NO.193 and a downstream primer as shown in SEQ ID NO. a downstream primer as shown in SEQ ID NO.194; an upstream primer as shown in SEQ ID NO.195 and a downstream primer as shown in SEQ ID NO.196; an upstream primer as shown in SEQ ID NO.197 and a downstream primer as shown in SEQ ID NO.198; an upstream primer as shown in SEQ ID NO.199 and a downstream primer as shown in SEQ ID NO.200; an upstream primer as shown in SEQ ID NO.201 and a downstream primer as shown in SEQ ID NO.202; an upstream primer as shown in SEQ ID NO.203 and a downstream primer as shown in SEQ ID NO.204; an upstream primer as shown in SEQ ID NO.205 and a downstream primer as shown in SEQ ID NO.206; an upstream primer as shown in SEQ ID NO.207 and a downstream primer as shown in SEQ ID NO.208; an upstream primer as shown in SEQ ID NO.209 and a downstream primer as shown in SEQ ID NO.210; a The upstream primer shown in SEQ ID NO.211 and the downstream primer shown in SEQ ID NO.212; the upstream primer shown in SEQ ID NO.213 and the downstream primer shown in SEQ ID NO.214; the upstream primer shown in SEQ ID NO.215 and the downstream primer shown in SEQ ID NO.216; the upstream primer shown in SEQ ID NO.217 and the downstream primer shown in SEQ ID NO.218; an upstream primer having a sequence as shown in SEQ ID NO.219 and a downstream primer having a sequence as shown in SEQ ID NO.220; an upstream primer having a sequence as shown in SEQ ID NO.221 and a downstream primer having a sequence as shown in SEQ ID NO.222; an upstream primer having a sequence as shown in SEQ ID NO.223 and a downstream primer having a sequence as shown in SEQ ID NO.224; an upstream primer having a sequence as shown in SEQ ID NO.225 and a downstream primer having a sequence as shown in SEQ ID NO.226; an upstream primer having a sequence as shown in SEQ ID NO.227 and a downstream primer having a sequence as shown in SEQ ID NO.228; an upstream primer having a sequence as shown in SEQ ID NO.229 and a downstream primer having a sequence as shown in SEQ ID NO.230; an upstream primer having a sequence as shown in SEQ ID NO.231 and a downstream primer having a sequence as shown in SEQ ID NO.232; an upstream primer having a sequence as shown in SEQ ID NO.233 and a downstream primer having a sequence as shown in SEQ ID NO.234; The upstream primer shown in NO.235 and the downstream primer whose sequence is shown in SEQ ID NO.236;.

[0022] The primer pair for detecting PTEN gene DNA mutation is selected from at least one of the following pairs: an upstream primer with a sequence as shown in SEQ ID NO.237 and a downstream primer with a sequence as shown in SEQ ID NO.238; an upstream primer with a sequence as shown in SEQ ID NO.239 and a downstream primer with a sequence as shown in SEQ ID NO.240; an upstream primer with a sequence as shown in SEQ ID NO.241 and a downstream primer with a sequence as shown in SEQ ID NO.242; an upstream primer with a sequence as shown in SEQ ID NO.243 and a downstream primer with a sequence as shown in SEQ ID NO.244; an upstream primer with a sequence as shown in SEQ ID NO.245 and a downstream primer with a sequence as shown in SEQ ID NO.246; an upstream primer with a sequence as shown in SEQ ID NO.247 and a downstream primer with a sequence as shown in SEQ ID NO.248; an upstream primer with a sequence as shown in SEQ ID NO.249 and a downstream primer with a sequence as shown in SEQ ID NO.250; an upstream primer with a sequence as shown in SEQ ID NO.251 and a downstream primer with a sequence as shown in SEQ ID NO.252; an upstream primer as shown in SEQ ID NO.253 and a downstream primer as shown in SEQ ID NO.254; an upstream primer as shown in SEQ ID NO.255 ​​and a downstream primer as shown in SEQ ID NO.256; an upstream primer as shown in SEQ ID NO.257 and a downstream primer as shown in SEQ ID NO.258; an upstream primer as shown in SEQ ID NO.259 and a downstream primer as shown in SEQ ID NO.260; an upstream primer as shown in SEQ ID NO.261 and a downstream primer as shown in SEQ ID NO.262; an upstream primer as shown in SEQ ID NO.263 and a downstream primer as shown in SEQ ID NO.264; an upstream primer as shown in SEQ ID NO.265 and a downstream primer as shown in SEQ ID NO.266; an upstream primer as shown in SEQ ID NO.267 and a downstream primer as shown in SEQ ID NO.268; an upstream primer as shown in SEQ ID NO.269 and a downstream primer as shown in SEQ ID NO. 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; 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 primeran upstream primer as shown in SEQ ID NO.277 and a downstream primer as shown in SEQ ID NO.278; an upstream primer as shown in SEQ ID NO.279 and a downstream primer as shown in SEQ ID NO.280; an upstream primer as shown in SEQ ID NO.281 and a downstream primer as shown in SEQ ID NO.282; an upstream primer as shown in SEQ ID NO.283 and a downstream primer as shown in SEQ ID NO.284; an upstream primer as shown in SEQ ID NO.285 and a downstream primer as shown in SEQ ID NO.286; an upstream primer as shown in SEQ ID NO.287 and a downstream primer as shown in SEQ ID NO.288; an upstream primer as shown in SEQ ID NO.289 and a downstream primer as shown in SEQ ID NO.290; an upstream primer as shown in SEQ ID NO.291 and a downstream primer as shown in SEQ ID NO.292; an upstream primer as shown in SEQ ID NO.293 and a downstream primer as shown in SEQ ID NO. NO.294; an upstream primer having a sequence as shown in SEQ ID NO.295 and a downstream primer having a sequence as shown in SEQ ID NO.296; an upstream primer having a sequence as shown in SEQ ID NO.297 and a downstream primer having a sequence as shown in SEQ ID NO.298; an upstream primer having a sequence as shown in SEQ ID NO.299 and a downstream primer having a sequence as shown in SEQ ID NO.300; an upstream primer having a sequence as shown in SEQ ID NO.301 and a downstream primer having a sequence as shown in SEQ ID NO.302; an upstream primer having a sequence as shown in SEQ ID NO.303 and a downstream primer having a sequence as shown in SEQ ID NO.304; an upstream primer having a sequence as shown in SEQ ID NO.305 and a downstream primer having a sequence as shown in SEQ ID NO.306; an upstream primer having a sequence as shown in SEQ ID NO.307 and a downstream primer having a sequence as shown in SEQ ID NO.308; an upstream primer having a sequence as shown in SEQ ID NO.309 and a downstream primer having a sequence as shown in SEQ ID NO.310; a The upstream primer shown in SEQ ID NO.311 and the downstream primer shown in SEQ ID NO.312; the upstream primer shown in SEQ ID NO.313 and the downstream primer shown in SEQ ID NO.314; 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 primeran upstream primer as shown in SEQ ID NO.319 and a downstream primer as shown in SEQ ID NO.320; an upstream primer as shown in SEQ ID NO.321 and a downstream primer as shown in SEQ ID NO.322; an upstream primer as shown in SEQ ID NO.323 and a downstream primer as shown in SEQ ID NO.324; an upstream primer as shown in SEQ ID NO.325 and a downstream primer as shown in SEQ ID NO.326; an upstream primer as shown in SEQ ID NO.327 and a downstream primer as shown in SEQ ID NO.328; an upstream primer as shown in SEQ ID NO.329 and a downstream primer as shown in SEQ ID NO.330; an upstream primer as shown in SEQ ID NO.331 and a downstream primer as shown in SEQ ID NO.332; an upstream primer as shown in SEQ ID NO.333 and a downstream primer as shown in SEQ ID NO.334; an upstream primer as shown in SEQ ID NO.335 and a downstream primer as shown in SEQ ID NO. NO.336; an upstream primer having a sequence as shown in SEQ ID NO.337 and a downstream primer having a sequence as shown in SEQ ID NO.338; an upstream primer having a sequence as shown in SEQ ID NO.339 and a downstream primer having a sequence as shown in SEQ ID NO.340; an upstream primer having a sequence as shown in SEQ ID NO.341 and a downstream primer having a sequence as shown in SEQ ID NO.342; an upstream primer having a sequence as shown in SEQ ID NO.343 and a downstream primer having a sequence as shown in SEQ ID NO.344; an upstream primer having a sequence as shown in SEQ ID NO.345 and a downstream primer having a sequence as shown in SEQ ID NO.346; an upstream primer having a sequence as shown in SEQ ID NO.347 and a downstream primer having a sequence as shown in SEQ ID NO.348; an upstream primer having a sequence as shown in SEQ ID NO.349 and a downstream primer having a sequence as shown in SEQ ID NO.350; an upstream primer having a sequence as shown in SEQ ID NO.351 and a downstream primer having a sequence as shown in SEQ ID NO.352; The upstream primer shown in NO.353 and the downstream primer whose sequence is shown in SEQ ID NO.354;.

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

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

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

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

[0027] The primer pair for detecting RAF1 gene DNA mutation 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 comprises primer pairs for detecting RNA fusion variations of ALK, BRAF, NTRK1, NTRK2, NTRK3, PPARG, RET and THADA genes;

[0029] The ALK gene RNA fusion variation includes the ALK gene and the EML4, KIF5B or TPM3 gene RNA fusion variation, and the primer pair includes: upstream primers with sequences as shown in SEQ ID NOs.551 to 556, SEQ ID NOs.561 to 566, and SEQ ID NOs.581 to 584, and downstream primers with sequences as shown in SEQ ID NOs.473 to 480;

[0030] The BRAF gene RNA fusion variation is an RNA fusion variation of the BRAF gene and the AKAP9 gene, and the primer pair comprises: an upstream primer having a sequence as shown in SEQ ID NOs.485 to 490 and a downstream primer having a sequence as shown in SEQ ID NOs.481 to 484 and SEQ ID NOs.589 to 590;

[0031] The NTRK1 gene RNA fusion variation is an RNA fusion variation of the NTRK1 gene and the TPM3 gene, and the primer pair comprises: an upstream primer having a sequence as shown in SEQ ID NOs.581 to 584 and a downstream primer having a sequence as shown in SEQ ID NOs.491 to 508;

[0032] The NTRK2 gene RNA fusion variation is an RNA fusion variation of the NTRK2 gene and the BCR gene, and the primer pair includes: an upstream primer having a sequence as shown in SEQ ID NOs.591 to 592 and a downstream primer having a sequence as shown in SEQ ID NOs.509 to 518;

[0033] The NTRK3 gene RNA fusion variation is an RNA fusion variation of the NTRK3 gene and the ETV6 gene, and the primer pair includes: upstream primers with sequences as shown in SEQ ID NOs.523-530 and SEQ ID NOs.557-560 and downstream primers with sequences as shown in SEQ ID NOs.519-522 and SEQ ID NOs.531-532;

[0034] The PPARG gene RNA fusion variation is an RNA fusion variation of the PPARG gene and the PAX8 gene, and the primer pair comprises: an upstream primer having a sequence as shown in SEQ ID NOs.573 to 578 and a downstream primer having a sequence as shown in SEQ ID NOs.587 to 588;

[0035] The RET gene RNA fusion variation includes the RNA fusion variation of the RET gene and the KIF5B, CCDC6, NCOA4 or PRKAR1A gene, and the primer pair includes: upstream primers with sequences as shown in SEQ ID NOs.561 to 566, SEQ ID NOs.545 to 550, SEQ ID NOs.567 to 572, and SEQ ID NOs.579 to 580, and downstream primers with sequences as shown in SEQ ID NOs.533 to 538;

[0036] The THADA gene RNA fusion variation is an RNA fusion variation of the THADA gene and the IGF2BP3 gene, and the primer pair includes: an upstream primer whose sequence is shown in SEQ ID NOs.539 to 544 and a downstream primer whose sequence is shown in SEQ ID NOs.585 to 586.

[0037] In some embodiments, the primer composition includes primer pairs for detecting DNA mutations in 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 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 a sequence as shown in SEQ ID NO.593 connected to their 5' ends, and a nucleotide fragment with a sequence as shown in SEQ ID NO.594 connected to their 3' ends.

[0039] Preferably, the 5' end of the upstream primer for detecting DNA mutations in AKT1, ARAF, BRAF, CDKN2A, CTNNB 1, DICER1, and GNAS genes and detecting RNA fusion variations in the BRAF gene is connected to a nucleotide fragment with a sequence as shown in SEQ ID NO.593, and the 3' end of the corresponding downstream primer is connected to a nucleotide fragment with a sequence as shown in SEQ ID NO.594.

[0040] The second aspect of the present invention provides the use of the primer combination as described above in the preparation of a product for detecting gene mutations associated with benign and malignant thyroid nodules.

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

[0042] The third aspect of the present invention provides a kit for detecting gene mutations associated with benign and malignant thyroid nodules, the kit comprising the primer combination as described above.

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

[0044] A fifth aspect of the present invention provides a system for detecting gene mutations associated with benign and 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 combination as described above or the kit as 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 connectors, aligning sequences to corresponding positions of reference genome data, annotating gene mutations and statistically analyzing corresponding data.

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

[0048] The present invention provides a primer composition for detecting DNA mutations (including single nucleotide variations (SNVs) and insertions and deletions (Indels) of small fragments) and RNA fusion variations of genes related to benign and malignant thyroid nodules. The primer pairs in the primer composition can be amplified separately, and can also perform high-efficiency, high-sensitivity and high-specificity amplification on DNA and cDNA templates to be tested under the same PCR reaction system and reaction procedure. The primers have no obvious interference with each other. One multiplex PCR amplification can quickly and accurately amplify 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 conditions of the related regions can be obtained, thereby assisting the clinical diagnosis of benign and malignant thyroid nodules.

[0049] Furthermore, in the present invention, a proper proportion of the upstream primer 5' end and the corresponding downstream primer 3' end in the primer composition are respectively connected to specific nucleotide sequences, and the nucleotide sequences will not affect the amplification effect of the primers in the primer composition, and can make the non-specific amplified fragments in the multiplex PCR amplification system form a "neck loop" structure, which cannot be used as a template for further amplification, thereby reducing the non-specific amplification in the multiplex PCR amplification system, further improving the uniformity and specificity of the multiplex PCR amplification, effectively reducing the amount of nucleic acid input detected by the primer composition of the present invention and reducing the minimum detection limit of the mutation frequency (which can be as low as 0.5%), and the number of detection reads is higher, so that the nucleic acid input of samples with a mutation frequency of more than 2% can be as low as 1ng, and the nucleic acid input of samples with a mutation frequency of 1% can be as low as 5ng. Therefore, the primer composition of the present invention is very suitable for related gene mutation detection in biological samples with small sample volume and low nucleic acid concentration, including thyroid fine needle aspiration samples, etc.

[0050] The detection method of the present invention can simultaneously detect gene variations at the DNA and RNA levels, is easy to operate, has low cost, can shorten the detection time to 4 days, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Flow chart of the detection method of the present invention. DETAILED DESCRIPTION

[0052] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0053] The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, such as the fourth edition of Molecular Cloning: A Laboratory Manual edited by Green and Sambrook, published in 2013, or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0054] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0055] In addition, as used in the present invention, the term "or" is an inclusive "or" symbol and is equivalent to the term "and / or", unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meanings of "a", "an", and "the" include plural referents. The meaning of "in..." includes "in..." and "on...".

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

[0057] Example 1

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

[0059] 1. Primer Composition

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

[0061] Table 1

[0062]

[0063]

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

[0065] Table 2 DNA mutation detection primers

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077] RNA fusion variation occurs on two genes, and the two genes fused are not fixed. The detection primers for fusion variation include upstream primers (for fusion upstream genes) and downstream primers (for fusion downstream genes). As shown in Table 1, ALK gene can undergo RNA fusion variation with EML4, KIF5B or TPM3 gene; BRAF gene can undergo RNA fusion variation with AKAP9 gene; NTRK1 gene can undergo RNA fusion variation with TPM3 gene; NTRK2 gene can undergo RNA fusion variation with BCR gene; NTRK3 gene can undergo RNA fusion variation with ETV6 gene; PPARG gene can undergo RNA fusion variation with PAX8 gene; RET gene can undergo RNA fusion variation with KIF5B, CCDC6, NCOA4 or PRKAR1A gene; THADA gene can undergo RNA fusion variation with IGF2BP3 gene. The design of the fusion variation detection primer of the present invention is to design primers at the position (fusion site) where the above-mentioned genes will undergo fusion, 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 an upstream gene of the fusion variant (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 related RNA fusion variations, 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 RNA fusion variation detection primers is the location information of the corresponding transcript in the genome, and the version number of the compared genome is hg19.

[0079] In Table 3, "Gene Name-F" means that the gene is located upstream of the fusion variation, and the upstream primer Forward Primer is designed for its fusion position; "Gene Name-R" means that the gene is located downstream of the fusion variation, and the downstream primer (Reverse Primer) is designed for its fusion position.

[0080] Table 3 Primers for RNA fusion mutation detection

[0081]

[0082]

[0083]

[0084]

[0085] The inventors further found that for the primers in Tables 2 and 3, when 50% to 100% (including 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 100%, 50% to 70%, 50% to 80%, 50% to 90%, 60% to 80%, 60% to 90%, 60% to 100%, 70% to 90%, 70% to 100%, 80% to 100%) of the upstream primers have a 5' end connection sequence such as the nucleotide fragment shown in SEQ ID NO.593 and the corresponding downstream primer has a 3' end connection sequence such as the nucleotide fragment shown in SEQ ID NO.594, it will not affect the amplification effect of the primers, and can effectively reduce nonspecific amplification in the multiplex PCR amplification system, further improving the uniformity and specificity of the multiplex PCR amplification. In this embodiment, the upstream primer (about 51%) for mutation detection of the gene shown in bold and underlined is preferably connected to a nucleotide fragment with a sequence as shown in SEQ ID NO.593 at its 5' end, and the corresponding downstream primer is connected to a nucleotide fragment with a sequence as shown in SEQ ID NO.594 at its 3' end.

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

[0087] 2. Test kit

[0088] The primers in Table 2 and Table 3 are mixed evenly to obtain a primer composition, wherein the final concentration of each primer in the primer composition ranges from 0.5 to 0.7 uM, and is preferably 0.6 uM in this embodiment; the kit comprises the primer composition.

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

[0090] Table 4

[0091]

[0092]

[0093] 3. Detection Methods

[0094] The detection method comprises the following steps:

[0095] Step 1: Sample preparation: Extract nucleic acid (DNA+RNA) from thyroid fine needle aspiration samples;

[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 multiple PCR amplification to obtain a round of amplicon products, purify the excess primers in the system, connect Index tags to both ends of the amplicon products to complete the construction of the amplicon library, perform library quality inspection and then sequence the library;

[0098] Step 4: Sequencing: Use Illumina NovaSeq 6000 sequencer (PE150) for sequencing;

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

[0100] Figure 1 It is a detection flow chart of the detection method of the present invention.

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

[0102] 1. cDNA Synthesis

[0103] 1.1 Take the components in kit 1 for the experiment, take samples according to the RNA input amount of 20-100ng and the DNA input amount of 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 components in the order of Table 5 below and set up the reaction system on an ice box. Since the extracted nucleic acid sample includes both DNA and RNA, the corresponding volume of DNA will be carried along with the addition of RNA in Table 5, which has no effect on the reverse transcription reaction. The DNA added to the subsequent multiple 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 ice.

[0108] 1.3 The mixed system in the PCR tube was shaken and mixed and centrifuged, and placed on a PCR instrument to perform the reaction as shown in Table 6 below:

[0109] Table 6

[0110] temperature Reaction time Number of cycles 50℃ 15min 1 85℃ 5s 1 8℃ Hold 1

[0111] 2. Purification after cDNA Synthesis

[0112] 2.1 Preparation before purification: Equilibration of magnetic beads (reagent 11): Take out 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] 2.2cDNA product purification:

[0114] 2.2.1 Mixing beads: Oscillate AMPure XP Beads until they are evenly mixed (Important note: AMPure beads must be mixed evenly before purification, otherwise it will affect the purification effect).

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

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

[0117] 2.2.4 Separation of magnetic beads: Place the PCR plate / tubes on a magnetic stand for 5 minutes until the solution becomes clear.

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

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

[0120] Important tips: Try to avoid contact between 70% ethanol and air. Otherwise, the concentration of ethanol will change, affecting the effect of washing magnetic beads. Prepare enough 70% ethanol each time.

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

[0122] 2.2.8 Remove the remaining ethanol: Completely remove the remaining traces of ethanol in each tube. Centrifuge briefly for 10-15 seconds, put the PCR plate / tube back into the magnetic stand, and use a 10-20μL pipette tip to remove the remaining ethanol solution at the bottom of the tube.

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

[0124] IMPORTANT: Do not over-dry the beads. When small cracks appear in the middle of the bead mass, the beads are sufficiently dry. If large cracks appear throughout the bead mass, or if the beads break into small flakes, the beads are over-dried. Over-dried beads will be difficult to resuspend.

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

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

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

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

[0129] 3.1 Take the components in kit 2 for the experiment, take 0.2 ml PCR tubes according to the number of samples, and mark the sample numbers on the tube caps.

[0130] 3.2 Add the components in the order of Table 7 below and set up 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 ice.

[0134] 3.3 The mixed system in the PCR tube was shaken and mixed and centrifuged, and placed on a PCR instrument to perform the reaction as 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. Place the gene-specific PCR product at room temperature:

[0140] 4.2 Dilute Reagent 8, according to the ratio of Reagent 8: Nuclease-free water = 3:2, dilute according to the number of samples, place the prepared reaction solution on ice, and configure the enzymatic system according to the reaction system in Table 9 below:

[0141] Table 9

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

[0143] 4.3 The mixed system in the PCR tube was shaken and centrifuged, and placed on a PCR instrument to perform the reaction as shown in Table 10 below:

[0144] Table 10

[0145] temperature Reaction time Number of cycles 37℃ 20min 1 80℃ 10min 1 8℃ Hold 1

[0146] Obtain gene-specific PCR products after enzymatic purification.

[0147] 5. Purification of Gene-specific PCR Products

[0148] 5.1 Preparation before purification: Equilibration of magnetic beads (reagent 11): Take out 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 Gene-specific PCR product purification:

[0150] 5.2.1 Mixing beads: Oscillate AMPure XP Beads until they are evenly mixed (Important note: AMPure beads must be mixed evenly before purification, otherwise it will affect the purification effect).

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

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

[0153] 5.2.4 Separation of magnetic beads: Place the PCR plate / tubes on a magnetic stand for 5 minutes until the solution becomes clear.

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

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

[0156] Important tips: Try to avoid contact between 70% ethanol and air. Otherwise, the concentration of ethanol will change, affecting the effect of washing magnetic beads. Prepare enough 70% ethanol each time.

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

[0158] 5.2.8 Remove the remaining ethanol: Completely remove the remaining traces of ethanol in each tube. Centrifuge briefly for 10-15 seconds, put the PCR plate / tube back into the magnetic stand, and use a 10-20μL pipette tip to remove the remaining ethanol solution at the bottom of the tube.

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

[0160] IMPORTANT: Do not over-dry the beads. When small cracks appear in the middle of the bead mass, the beads are sufficiently dry. If large cracks appear throughout the bead mass, or if the beads break into small flakes, the beads are over-dried. Over-dried beads will be difficult to resuspend.

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

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

[0163] 5.2.12 Separate the supernatant: Place the PCR plate / tube back on the magnetic rack until the solution becomes clear, which may take 5 minutes. Pipette 18 μL of the supernatant into a new PCR tube (pause point: can be stored at -20°C) to obtain the gene-specific PCR purification product.

[0164] 6. Indexing PCR (library amplification)

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

[0166] The reaction solution is prepared according to the system in Table 11:

[0167] Table 11

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

[0169] 6.2 The mixed system in the PCR tube was shaken and mixed and centrifuged, and placed on a PCR instrument to perform the reaction as shown in Table 12 below:

[0170] Table 12

[0171]

[0172] Obtain an amplicon library.

[0173] 7. Library purification:

[0174] 7.1 Preparation before purification: Equilibration of magnetic beads (reagent 11): Take out 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.

[0175] 7.2 Library purification:

[0176] 7.2.1 Mixing magnetic beads: Oscillate AMPure XP Beads until they are evenly mixed (Important note: AMPure beads must be mixed evenly before purification, otherwise it will affect the purification effect).

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

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

[0179] 7.2.4 Separation of magnetic beads: Place the PCR plate / tubes on a magnetic stand for 5 minutes until the solution becomes clear.

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

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

[0182] Important tips: Try to avoid contact between 70% ethanol and air. Otherwise, the concentration of ethanol will change, affecting the effect of washing magnetic beads. Prepare enough 70% ethanol each time.

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

[0184] 7.2.8 Remove the remaining ethanol: Completely remove the remaining traces of ethanol in each tube. Centrifuge briefly for 10-15 seconds, put the PCR plate / tube back into the magnetic stand, and use a 10-20μL pipette tip to remove the remaining ethanol solution at the bottom of the tube.

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

[0186] IMPORTANT: Do not over-dry the beads. When small cracks appear in the middle of the bead mass, the beads are sufficiently dry. If large cracks appear throughout the bead mass, or if the beads break into small flakes, the beads are over-dried. Over-dried beads will be difficult to resuspend.

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

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

[0189] 7.2.12 Separate the supernatant: Place the PCR plate / tube back on the magnetic stand until the solution becomes clear, which may take 5 minutes. Pipette 20 μL of the supernatant into a new PCR tube (pause point: can be stored at -20°C) to obtain the purified amplicon library.

[0190] 8. Library quality control

[0191] 8.1Qubit quantification: Take 1μL library and use Qubit to detect library concentration. Library quality inspection standard: concentration greater than 1ng / ul, total amount not less than 20ng.

[0192] 8.2Qseq detected the library length, and the main peak was around 400b.

[0193] 9. Sequencing

[0194] Use the Illumina sequencing platform and NovaSeq 6000 sequencer for sequencing. It is recommended to use the Novasek 6000S1 Reagent Kit v1.5 (PE150) sequencing chip for sequencing. 1G data volume is required for each sample.

[0195] 10. Bioinformatics automation 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 Remove the connector Sentieon v202112.04 Reads mapping Sequence alignment annovar v2020 SNV / Indel annotation Annotation of SNV / Indel variants STAR-Fusion v1.10.0 Gene Fusion Calling Detection of gene fusion variants Arriba v2.3.0 Gene Fusion Calling Detection of gene fusion variants VarDict 1.8.3 SNV / Indel Calling Detect SNV / Indel mutations

[0199] 10.2 Use Snakemake language rules to write rules programs. Use graphviz to generate a process run logic diagram, and use the -dry-run mode to check whether the program logic is correct. After the check is completed, run the program, 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 data are as follows: Taking into account the actual sample sequencing quality and the requirement for the variation detection software to accurately detect variations, 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) The average number of times each base is sequenced ≥3000X >500x base ratio The percentage of bases sequenced more than 500 times ≥85％ Sequence alignment rate (On Target) How well the sequencing results match 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 the detection method were the same as those in Example 1.

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

[0206] Table 15

[0207]

[0208]

[0209] The FFPE standard products were sequenced and tested, 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 was in line with the expected results.

[0210] 2. SNV / Indel accuracy verification of clinical samples with known gene mutation results (hybridization capture sequencing): 200 ng of DNA samples were tested, and the accuracy comparison results were shown in Table 16 below.

[0211] Table 16

[0212]

[0213]

[0214] 21 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 detection kit of the present invention is tested for repeatability, and the kit and the detection method are the same as those in Example 1.

[0217] 1. By performing batch-to-batch and batch-to-batch consistency tests on FFPE standards (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 in the same batch, and SNV / Indel were consistently detected, with an intra-batch consistency of 100%.

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

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

[0223] Example 4

[0224] The detection kit of the present invention is used to detect the minimum nucleic acid input amount for verification, and the kit and the detection method are the same as those in Example 1.

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

[0226] Table 18

[0227]

[0228] The FFPE standards were sequenced and tested with different input amounts. Under the conditions of five DNA input amounts (5ng, 10ng, 25ng, 50ng, and 100ng), two 1% SNV sites were stably detected. Therefore, the detection kit of the present invention can still stably detect 1% SNV sites when the DNA input amount is as low as 5ng.

[0229] Further testing was performed with lower DNA input amounts.

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

[0231] Table 19

[0232]

[0233] The results showed that when the FFPE standard was subjected to lower DNA input (3ng, 2ng, 1ng), more than 2% of SNV sites could still be stably detected when the DNA input was as low as 1ng.

[0234] Example 5

[0235] The detection kit of the present invention is used to verify the minimum detection limit, and the kit and the detection method are the same as those in Example 1.

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

[0237] Table 20

[0238]

[0239]

[0240] The minimum detection limit of the ctDNA standard with a mutation frequency of 0.5% was verified. 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 detection mutation frequency of this kit can be as low as 0.5%.

[0241] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A primer composition for detecting gene mutations associated with benign and malignant thyroid nodules, characterized in that: Includes primer pairs for detecting DNA mutations in the BRAF and KRAS genes; The primer pair for detecting BRAF gene DNA mutation is selected from at least one of the following pairs: an upstream primer with a sequence as shown in SEQ ID NO.5 and a downstream primer with a sequence as shown in SEQ ID NO.6; an upstream primer with a sequence as shown in SEQ ID NO.7 and a downstream primer with a sequence as shown in SEQ ID NO.8; an upstream primer with a sequence as shown in SEQ ID NO.9 and a downstream primer with a sequence as shown in SEQ ID NO.10; The primer pair for detecting KRAS gene DNA mutation is selected from at least one of the following pairs: an upstream primer with a sequence as shown in SEQ ID NO.121 and a downstream primer with a sequence as shown in SEQ ID NO.122; an upstream primer with a sequence as shown in SEQ ID NO.123 and a downstream primer with a sequence as shown in SEQ ID NO.124; an upstream primer with a sequence as shown in SEQ ID NO.125 and a downstream primer with a sequence as shown in SEQ ID NO.126; an upstream primer with a sequence as shown in SEQ ID NO.127 and a downstream primer with a sequence as shown in SEQ ID NO.

128.

2. The primer composition according to claim 1, characterized in that Also included is at least one of the primer pairs for detecting DNA mutations in NRAS, TERT, TP53, and RET genes; The primer pair for detecting NRAS gene DNA mutation is selected from at least one of the following pairs: an upstream primer with a sequence as shown in SEQ ID NO.129 and a downstream primer with a sequence as shown in SEQ ID NO.130; an upstream primer with a sequence as shown in SEQ ID NO.131 and a downstream primer with a sequence as shown in SEQ ID NO.132; an upstream primer with a sequence as shown in SEQ ID NO.133 and a downstream primer with a sequence as shown in SEQ ID NO.134; an upstream primer with a sequence as shown in SEQ ID NO.135 and a downstream primer with a sequence as shown in SEQ ID NO.136; The primer pair for detecting TERT gene DNA mutation includes: an upstream primer with a sequence as shown in SEQ ID NO.385 and a downstream primer with a sequence as shown in SEQ ID NO.386; The primer pair for detecting TP53 gene DNA mutation is selected from at least one of the following pairs: an upstream primer with a sequence as shown in SEQ ID NO.387 and a downstream primer with a sequence as shown in SEQ ID NO.388; an upstream primer with a sequence as shown in SEQ ID NO.389 and a downstream primer with a sequence as shown in SEQ ID NO.390; an upstream primer with a sequence as shown in SEQ ID NO.391 and a downstream primer with a sequence as shown in SEQ ID NO.392; an upstream primer with a sequence as shown in SEQ ID NO.393 and a downstream primer with a sequence as shown in SEQ ID NO.394; an upstream primer with a sequence as shown in SEQ ID NO.395 and a downstream primer with a sequence as shown in SEQ ID NO.396; an upstream primer with a sequence as shown in SEQ ID NO.397 and a downstream primer with a sequence as shown in SEQ ID NO.398; an upstream primer with a sequence as shown in SEQ ID NO.399 and a downstream primer with a sequence as shown in SEQ ID NO.400; an upstream primer with a sequence as shown in SEQ ID NO.401 and a downstream primer with a sequence as shown in SEQ ID NO.402; An upstream primer as shown in NO.403 and a downstream primer as shown in SEQ ID NO.404; The primer pair for detecting RET gene DNA mutation is selected from at least one of the following pairs: an upstream primer with a sequence as shown in SEQ ID NO.357 and a downstream primer with a sequence as shown in SEQ ID NO.358; an upstream primer with a sequence as shown in SEQ ID NO.359 and a downstream primer with a sequence as shown in SEQ ID NO.360; an upstream primer with a sequence as shown in SEQ ID NO.361 and a downstream primer with a sequence as shown in SEQ ID NO.362; an upstream primer with a sequence as shown in SEQ ID NO.363 and a downstream primer with a sequence as shown in SEQ ID NO.364; an upstream primer with a sequence as shown in SEQ ID NO.365 and a downstream primer with a sequence as shown in SEQ ID NO.366; an upstream primer with a sequence as shown in SEQ ID NO.367 and a downstream primer with a sequence as shown in SEQ ID NO.368; an upstream primer with a sequence as shown in SEQ ID NO.369 and a downstream primer with a sequence as shown in SEQ ID NO.370; an upstream primer with a sequence as shown in SEQ ID NO.371 and a downstream primer with a sequence as shown in SEQ ID NO.372; The upstream primer shown in 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.

3. The primer composition according to claim 2, characterized in that Also included is at least one of the primer pairs for detecting DNA mutations in the CDKN2A, DICER1, EIF1AX, GNAS, HRAS, PIK3CA, PTEN, TSHR, ARAF, AKT1, CTNNB1, and RAF1 genes; The primer pair for detecting the DNA mutation of the CDKN2A gene is selected from at least one of the following pairs: an upstream primer having a sequence as shown in SEQ ID NO.11 and a downstream primer having a sequence as shown in SEQ ID NO.12; an upstream primer having a sequence as shown in SEQ ID NO.13 and a downstream primer having a sequence as shown in SEQ ID NO.14; an upstream primer having a sequence as shown in SEQ ID NO.15 and a downstream primer having a sequence as shown in SEQ ID NO.16; an upstream primer having a sequence as shown in SEQ ID NO.17 and a downstream primer having a sequence as shown in SEQ ID NO.18; an upstream primer having a sequence as shown in SEQ ID NO.19 and a downstream primer having a sequence as shown in SEQ ID NO.20; an upstream primer having a sequence as shown in SEQ ID NO.21 and a downstream primer having a sequence as shown in SEQ ID NO.22; an upstream primer having a sequence as shown in SEQ ID NO.23 and a downstream primer having a sequence as shown in SEQ ID NO.24; an upstream primer having a sequence as shown in SEQ ID NO.25 and a downstream primer having a sequence as shown in SEQ ID NO.26; an upstream primer having a sequence as shown in SEQ ID NO.27 and a downstream primer having a sequence as shown in SEQ ID NO. A downstream primer as shown in SEQ ID NO.28; an upstream primer as shown in SEQ ID NO.29 and a downstream primer as shown in SEQ ID NO.30; an upstream primer as shown in SEQ ID NO.31 and a downstream primer as shown in SEQ ID NO.32; The primer pair for detecting DNA mutation of DICER1 gene is selected from at least one of the following pairs: an upstream primer having a sequence as shown in SEQ ID NO.41 and a downstream primer having a sequence as shown in SEQ ID NO.42; an upstream primer having a sequence as shown in SEQ ID NO.43 and a downstream primer having a sequence as shown in SEQ ID NO.44; an upstream primer having a sequence as shown in SEQ ID NO.45 and a downstream primer having a sequence as shown in SEQ ID NO.46; The primer pair for detecting the EIF1AX gene DNA mutation is selected from at least one of the following pairs: an upstream primer having a sequence as shown in SEQ ID NO.47 and a downstream primer having a sequence as shown in SEQ ID NO.48; an upstream primer having a sequence as shown in SEQ ID NO.49 and a downstream primer having a sequence as shown in SEQ ID NO.50; an upstream primer having a sequence as shown in SEQ ID NO.51 and a downstream primer having a sequence as shown in SEQ ID NO.52; an upstream primer having a sequence as shown in SEQ ID NO.53 and a downstream primer having a sequence as shown in SEQ ID NO.54; an upstream primer having a sequence as shown in SEQ ID NO.55 and a downstream primer having a sequence as shown in SEQ ID NO.56; an upstream primer having a sequence as shown in SEQ ID NO.57 and a downstream primer having a sequence as shown in SEQ ID NO.58; an upstream primer having a sequence as shown in SEQ ID NO.59 and a downstream primer having a sequence as shown in SEQ ID NO.60; an upstream primer having a sequence as shown in SEQ ID NO.61 and a downstream primer having a sequence as shown in SEQ ID NO.62; an upstream primer having a sequence as shown in SEQ ID NO.63 and a downstream primer having a sequence as shown in SEQ ID NO. NO.64; an upstream primer having a sequence as shown in SEQ ID NO.65 and a downstream primer having a sequence as shown in SEQ ID NO.66; an upstream primer having a sequence as shown in SEQ ID NO.67 and a downstream primer having a sequence as shown in SEQ ID NO.68; an upstream primer having a sequence as shown in SEQ ID NO.69 and a downstream primer having a sequence as shown in SEQ ID NO.70; an upstream primer having a sequence as shown in SEQ ID NO.71 and a downstream primer having a sequence as shown in SEQ ID NO.72; an upstream primer having a sequence as shown in SEQ ID NO.73 and a downstream primer having a sequence as shown in SEQ ID NO.74; an upstream primer having a sequence as shown in SEQ ID NO.75 and a downstream primer having a sequence as shown in SEQ ID NO.76; an upstream primer having a sequence as shown in SEQ ID NO.77 and a downstream primer having a sequence as shown in SEQ ID NO.78; an upstream primer having a sequence as shown in SEQ ID NO.79 and a downstream primer having a sequence as shown in SEQ ID NO.80; an upstream primer having a sequence as shown in SEQ ID NO.81 and a downstream primer having a sequence as 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 upstream primer shown in SEQ ID NO.87 and the downstream primer shown in SEQ ID NO.88; an upstream primer having a sequence as shown in SEQ ID NO.89 and a downstream primer having a sequence as shown in SEQ ID NO.90; an upstream primer having a sequence as shown in SEQ ID NO.91 and a downstream primer having a sequence as shown in SEQ ID NO.92; an upstream primer having a sequence as shown in SEQ ID NO.93 and a downstream primer having a sequence as shown in SEQ ID NO.94; an upstream primer having a sequence as shown in SEQ ID NO.95 and a downstream primer having a sequence as shown in SEQ ID NO.96; an upstream primer having a sequence as shown in SEQ ID NO.97 and a downstream primer having a sequence as shown in SEQ ID NO.98: an upstream primer having a sequence as shown in SEQ ID NO.99 and a downstream primer having a sequence as shown in SEQ ID NO.100; an upstream primer having a sequence as shown in SEQ ID NO.101 and a downstream primer having a sequence as shown in SEQ ID NO.102; an upstream primer having a sequence as shown in SEQ ID NO.103 and a downstream primer having a sequence as shown in SEQ ID NO.104; an upstream primer having a sequence as shown in SEQ ID NO.105 and a downstream primer having a sequence as shown in SEQ ID NO. A downstream primer as shown in SEQ ID NO.106; an upstream primer as shown in SEQ ID NO.107 and a downstream primer as shown in SEQ ID NO.108;. The primer pair for detecting the GNAS gene DNA mutation is selected from at least one of the following pairs: an upstream primer having a sequence as shown in SEQ ID NO.109 and a downstream primer having a sequence as shown in SEQ ID NO.110; an upstream primer having a sequence as shown in SEQ ID NO.111 and a downstream primer having a sequence as shown in SEQ ID NO.112; The primer pair for detecting HRAS gene DNA mutation is selected from at least one of the following pairs: an upstream primer with a sequence as shown in SEQ ID NO.113 and a downstream primer with a sequence as shown in SEQ ID NO.114; an upstream primer with a sequence as shown in SEQ ID NO.115 and a downstream primer with a sequence as shown in SEQ ID NO.116; an upstream primer with a sequence as shown in SEQ ID NO.117 and a downstream primer with a sequence as shown in SEQ ID NO.118; an upstream primer with a sequence as shown in SEQ ID NO.119 and a downstream primer with a sequence as shown in SEQ ID NO.120; The primer pair for detecting the PIK3CA gene DNA mutation is selected from at least one of the following pairs: an upstream primer with a sequence as shown in SEQ ID NO.137 and a downstream primer with a sequence as shown in SEQ ID NO.138; an upstream primer with a sequence as shown in SEQ ID NO.139 and a downstream primer with a sequence as shown in SEQ ID NO.140; an upstream primer with a sequence as shown in SEQ ID NO.141 and a downstream primer with a sequence as shown in SEQ ID NO.142; an upstream primer with a sequence as shown in SEQ ID NO.143 and a downstream primer with a sequence as shown in SEQ ID NO.144; an upstream primer with a sequence as shown in SEQ ID NO.145 and a downstream primer with a sequence as shown in SEQ ID NO.146; an upstream primer with a sequence as shown in SEQ ID NO.147 and a downstream primer with a sequence as shown in SEQ ID NO.148; an upstream primer with a sequence as shown in SEQ ID NO.149 and a downstream primer with a sequence as shown in SEQ ID NO.150; an upstream primer with a sequence as shown in SEQ ID NO.151 and a downstream primer with a sequence as shown in SEQ ID NO.152; an upstream primer as shown in SEQ ID NO.153 and a downstream primer as shown in SEQ ID NO.154; an upstream primer as shown in SEQ ID NO.155 and a downstream primer as shown in SEQ ID NO.156; an upstream primer as shown in SEQ ID NO.157 and a downstream primer as shown in SEQ ID NO.158; an upstream primer as shown in SEQ ID NO.159 and a downstream primer as shown in SEQ ID NO.160; an upstream primer as shown in SEQ ID NO.161 and a downstream primer as shown in SEQ ID NO.162; an upstream primer as shown in SEQ ID NO.163 and a downstream primer as shown in SEQ ID NO.164; an upstream primer as shown in SEQ ID NO.165 and a downstream primer as shown in SEQ ID NO.166; an upstream primer as shown in SEQ ID NO.167 and a downstream primer as shown in SEQ ID NO.168; an upstream primer as shown in SEQ ID NO.169 and a downstream primer as shown in SEQ ID NO.170; NO.170; an upstream primer having a sequence as shown in SEQ ID NO.171 and a downstream primer having a sequence as shown in SEQ ID NO.172; an upstream primer having a sequence as shown in SEQ ID NO.173 and a downstream primer having a sequence as shown in SEQ ID NO.174; an upstream primer having a sequence as shown in SEQ ID NO.175 and a downstream primer having a sequence as shown in SEQ ID NO.176; a downstream primer having a sequence as shown in SEQ ID NO.an upstream primer as shown in SEQ ID NO.177 and a downstream primer as shown in SEQ ID NO.178; an upstream primer as shown in SEQ ID NO.179 and a downstream primer as shown in SEQ ID NO.180; an upstream primer as shown in SEQ ID NO.181 and a downstream primer as shown in SEQ ID NO.182; an upstream primer as shown in SEQ ID NO.183 and a downstream primer as shown in SEQ ID NO.184; an upstream primer as shown in SEQ ID NO.185 and a downstream primer as shown in SEQ ID NO.186; an upstream primer as shown in SEQ ID NO.187 and a downstream primer as shown in SEQ ID NO.188; an upstream primer as shown in SEQ ID NO.189 and a downstream primer as shown in SEQ ID NO.190; an upstream primer as shown in SEQ ID NO.191 and a downstream primer as shown in SEQ ID NO.192; an upstream primer as shown in SEQ ID NO.193 and a downstream primer as shown in SEQ ID NO. a downstream primer as shown in SEQ ID NO.194; an upstream primer as shown in SEQ ID NO.195 and a downstream primer as shown in SEQ ID NO.196; an upstream primer as shown in SEQ ID NO.197 and a downstream primer as shown in SEQ ID NO.198; an upstream primer as shown in SEQ ID NO.199 and a downstream primer as shown in SEQ ID NO.200; an upstream primer as shown in SEQ ID NO.201 and a downstream primer as shown in SEQ ID NO.202; an upstream primer as shown in SEQ ID NO.203 and a downstream primer as shown in SEQ ID NO.204; an upstream primer as shown in SEQ ID NO.205 and a downstream primer as shown in SEQ ID NO.206; an upstream primer as shown in SEQ ID NO.207 and a downstream primer as shown in SEQ ID NO.208; an upstream primer as shown in SEQ ID NO.209 and a downstream primer as shown in SEQ ID NO.210; a The upstream primer shown in SEQ ID NO.211 and the downstream primer shown in SEQ ID NO.212; the upstream primer shown in SEQ ID NO.213 and the downstream primer shown in SEQ ID NO.214; the upstream primer shown in SEQ ID NO.215 and the downstream primer shown in SEQ ID NO.216; the upstream primer shown in SEQ ID NO.217 and 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 primeran upstream primer as shown in SEQ ID NO.219 and a downstream primer as shown in SEQ ID NO.220; an upstream primer as shown in SEQ ID NO.221 and a downstream primer as shown in SEQ ID NO.222; an upstream primer as shown in SEQ ID NO.223 and a downstream primer as shown in SEQ ID NO.224; an upstream primer as shown in SEQ ID NO.225 and a downstream primer as shown in SEQ ID NO.226; an upstream primer as shown in SEQ ID NO.227 and a downstream primer as shown in SEQ ID NO.228; an upstream primer as shown in SEQ ID NO.229 and a downstream primer as shown in SEQ ID NO.230; an upstream primer as shown in SEQ ID NO.231 and a downstream primer as shown in SEQ ID NO.232; an upstream primer as shown in SEQ ID NO.233 and a downstream primer as shown in SEQ ID NO.234; an upstream primer as shown in SEQ ID NO.235 and a downstream primer as shown in SEQ ID NO. The downstream primer shown in NO.236;. The primer pair for detecting PTEN gene DNA mutation is selected from at least one of the following pairs: an upstream primer with a sequence as shown in SEQ ID NO.237 and a downstream primer with a sequence as shown in SEQ ID NO.238; an upstream primer with a sequence as shown in SEQ ID NO.239 and a downstream primer with a sequence as shown in SEQ ID NO.240; an upstream primer with a sequence as shown in SEQ ID NO.241 and a downstream primer with a sequence as shown in SEQ ID NO.242; an upstream primer with a sequence as shown in SEQ ID NO.243 and a downstream primer with a sequence as shown in SEQ ID NO.244; an upstream primer with a sequence as shown in SEQ ID NO.245 and a downstream primer with a sequence as shown in SEQ ID NO.246; an upstream primer with a sequence as shown in SEQ ID NO.247 and a downstream primer with a sequence as shown in SEQ ID NO.248; an upstream primer with a sequence as shown in SEQ ID NO.249 and a downstream primer with a sequence as shown in SEQ ID NO.250; an upstream primer with a sequence as shown in SEQ ID NO.251 and a downstream primer with a sequence as shown in SEQ ID NO.252; an upstream primer as shown in SEQ ID NO.253 and a downstream primer as shown in SEQ ID NO.254; an upstream primer as shown in SEQ ID NO.255 ​​and a downstream primer as shown in SEQ ID NO.256; an upstream primer as shown in SEQ ID NO.257 and a downstream primer as shown in SEQ ID NO.258; an upstream primer as shown in SEQ ID NO.259 and a downstream primer as shown in SEQ ID NO.260; an upstream primer as shown in SEQ ID NO.261 and a downstream primer as shown in SEQ ID NO.262; an upstream primer as shown in SEQ ID NO.263 and a downstream primer as shown in SEQ ID NO.264; an upstream primer as shown in SEQ ID NO.265 and a downstream primer as shown in SEQ ID NO.266; an upstream primer as shown in SEQ ID NO.267 and a downstream primer as shown in SEQ ID NO.268; an upstream primer as shown in SEQ ID NO.269 and a downstream primer as shown in SEQ ID NO. NO.270; an upstream primer having a sequence as shown in SEQ ID NO.271 and a downstream primer having a sequence as shown in SEQ ID NO.272; an upstream primer having a sequence as shown in SEQ ID NO.273 and a downstream primer having a sequence as shown in SEQ ID NO.274; an upstream primer having a sequence as shown in SEQ ID NO.275 and a downstream primer having a sequence as shown in SEQ ID NO.276; a downstream primer having a sequence as shown in SEQ ID NO.an upstream primer as shown in SEQ ID NO.277 and a downstream primer as shown in SEQ ID NO.278; an upstream primer as shown in SEQ ID NO.279 and a downstream primer as shown in SEQ ID NO.280; an upstream primer as shown in SEQ ID NO.281 and a downstream primer as shown in SEQ ID NO.282; an upstream primer as shown in SEQ ID NO.283 and a downstream primer as shown in SEQ ID NO.284; an upstream primer as shown in SEQ ID NO.285 and a downstream primer as shown in SEQ ID NO.286; an upstream primer as shown in SEQ ID NO.287 and a downstream primer as shown in SEQ ID NO.288; an upstream primer as shown in SEQ ID NO.289 and a downstream primer as shown in SEQ ID NO.290; an upstream primer as shown in SEQ ID NO.291 and a downstream primer as shown in SEQ ID NO.292; an upstream primer as shown in SEQ ID NO.293 and a downstream primer as shown in SEQ ID NO. NO.294; an upstream primer having a sequence as shown in SEQ ID NO.295 and a downstream primer having a sequence as shown in SEQ ID NO.296; an upstream primer having a sequence as shown in SEQ ID NO.297 and a downstream primer having a sequence as shown in SEQ ID NO.298; an upstream primer having a sequence as shown in SEQ ID NO.299 and a downstream primer having a sequence as shown in SEQ ID NO.300; an upstream primer having a sequence as shown in SEQ ID NO.301 and a downstream primer having a sequence as shown in SEQ ID NO.302; an upstream primer having a sequence as shown in SEQ ID NO.303 and a downstream primer having a sequence as shown in SEQ ID NO.304; an upstream primer having a sequence as shown in SEQ ID NO.305 and a downstream primer having a sequence as shown in SEQ ID NO.306; an upstream primer having a sequence as shown in SEQ ID NO.307 and a downstream primer having a sequence as shown in SEQ ID NO.308; an upstream primer having a sequence as shown in SEQ ID NO.309 and a downstream primer having a sequence as shown in SEQ ID NO.310; a The upstream primer shown in SEQ ID NO.311 and the downstream primer shown in SEQ ID NO.312; the upstream primer shown in SEQ ID NO.313 and the downstream primer shown in SEQ ID NO.314; 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 primeran upstream primer as shown in SEQ ID NO.319 and a downstream primer as shown in SEQ ID NO.320; an upstream primer as shown in SEQ ID NO.321 and a downstream primer as shown in SEQ ID NO.322; an upstream primer as shown in SEQ ID NO.323 and a downstream primer as shown in SEQ ID NO.324; an upstream primer as shown in SEQ ID NO.325 and a downstream primer as shown in SEQ ID NO.326; an upstream primer as shown in SEQ ID NO.327 and a downstream primer as shown in SEQ ID NO.328; an upstream primer as shown in SEQ ID NO.329 and a downstream primer as shown in SEQ ID NO.330; an upstream primer as shown in SEQ ID NO.331 and a downstream primer as shown in SEQ ID NO.332; an upstream primer as shown in SEQ ID NO.333 and a downstream primer as shown in SEQ ID NO.334; an upstream primer as shown in SEQ ID NO.335 and a downstream primer as shown in SEQ ID NO. NO.336; an upstream primer having a sequence as shown in SEQ ID NO.337 and a downstream primer having a sequence as shown in SEQ ID NO.338; an upstream primer having a sequence as shown in SEQ ID NO.339 and a downstream primer having a sequence as shown in SEQ ID NO.340; an upstream primer having a sequence as shown in SEQ ID NO.341 and a downstream primer having a sequence as shown in SEQ ID NO.342; an upstream primer having a sequence as shown in SEQ ID NO.343 and a downstream primer having a sequence as shown in SEQ ID NO.344; an upstream primer having a sequence as shown in SEQ ID NO.345 and a downstream primer having a sequence as shown in SEQ ID NO.346; an upstream primer having a sequence as shown in SEQ ID NO.347 and a downstream primer having a sequence as shown in SEQ ID NO.348; an upstream primer having a sequence as shown in SEQ ID NO.349 and a downstream primer having a sequence as shown in SEQ ID NO.350; an upstream primer having a sequence as shown in SEQ ID NO.351 and a downstream primer having a sequence as shown in SEQ ID NO.352; The upstream primer shown in NO.353 and the downstream primer whose sequence is shown in SEQ ID NO.354;. The primer pair for detecting TSHR gene DNA mutation is selected from at least one of the following pairs: an upstream primer with a sequence as shown in SEQ ID NO.405 and a downstream primer with a sequence as shown in SEQ ID NO.406; an upstream primer with a sequence as shown in SEQ ID NO.407 and a downstream primer with a sequence as shown in SEQ ID NO.408; an upstream primer with a sequence as shown in SEQ ID NO.409 and a downstream primer with a sequence as shown in SEQ ID NO.410; an upstream primer with a sequence as shown in SEQ ID NO.411 and a downstream primer with a sequence as shown in SEQ ID NO.412; an upstream primer with a sequence as shown in SEQ ID NO.413 and a downstream primer with a sequence as shown in SEQ ID NO.414; an upstream primer with a sequence as shown in SEQ ID NO.415 and a downstream primer with a sequence as shown in SEQ ID NO.416; an upstream primer with a sequence as shown in SEQ ID NO.417 and a downstream primer with a sequence as shown in SEQ ID NO.418; an upstream primer with a sequence as shown in SEQ ID NO.419 and a downstream primer with a sequence as shown in SEQ ID NO.420; an upstream primer as shown in SEQ ID NO.421 and a downstream primer as shown in SEQ ID NO.422; an upstream primer as shown in SEQ ID NO.423 and a downstream primer as shown in SEQ ID NO.424; an upstream primer as shown in SEQ ID NO.425 and a downstream primer as shown in SEQ ID NO.426; an upstream primer as shown in SEQ ID NO.427 and a downstream primer as shown in SEQ ID NO.428; an upstream primer as shown in SEQ ID NO.429 and a downstream primer as shown in SEQ ID NO.430; an upstream primer as shown in SEQ ID NO.431 and a downstream primer as shown in SEQ ID NO.432; an upstream primer as shown in SEQ ID NO.433 and a downstream primer as shown in SEQ ID NO.434; an upstream primer as shown in SEQ ID NO.435 and a downstream primer as shown in SEQ ID NO.436; an upstream primer as shown in SEQ ID NO.437 and a downstream primer as shown in SEQ ID NO. NO.438; an upstream primer having a sequence as shown in SEQ ID NO.439 and a downstream primer having a sequence as shown in SEQ ID NO.440; an upstream primer having a sequence as shown in SEQ ID NO.441 and a downstream primer having a sequence as shown in SEQ ID NO.442; an upstream primer having a sequence as shown in SEQ ID NO.443 and a downstream primer having a sequence as shown in SEQ ID NO.444; a downstream primer having a sequence as shown in SEQ ID NO.an upstream primer as shown in SEQ ID NO.445 and a downstream primer as shown in SEQ ID NO.446; an upstream primer as shown in SEQ ID NO.447 and a downstream primer as shown in SEQ ID NO.448; an upstream primer as shown in SEQ ID NO.449 and a downstream primer as shown in SEQ ID NO.450; an upstream primer as shown in SEQ ID NO.451 and a downstream primer as shown in SEQ ID NO.452; an upstream primer as shown in SEQ ID NO.453 and a downstream primer as shown in SEQ ID NO.454; an upstream primer as shown in SEQ ID NO.455 and a downstream primer as shown in SEQ ID NO.456; an upstream primer as shown in SEQ ID NO.457 and a downstream primer as shown in SEQ ID NO.458; an upstream primer as shown in SEQ ID NO.459 and a downstream primer as shown in SEQ ID NO.460; an upstream primer as shown in SEQ ID NO.461 and a downstream primer as shown in SEQ ID NO. NO.462; an upstream primer having a sequence as shown in SEQ ID NO.463 and a downstream primer having a sequence as shown in SEQ ID NO.464; an upstream primer having a sequence as shown in SEQ ID NO.465 and a downstream primer having a sequence as shown in SEQ ID NO.466; an upstream primer having a sequence as shown in SEQ ID NO.467 and a downstream primer having a sequence as shown in SEQ ID NO.468; an upstream primer having a sequence as shown in SEQ ID NO.469 and a downstream primer having a sequence as shown in SEQ ID NO.470; an upstream primer having a sequence as shown in SEQ ID NO.471 and a downstream primer having a sequence as shown in SEQ ID NO.472;. The primer pair for detecting ARAF gene DNA mutation includes: an upstream primer with a sequence as shown in SEQ ID NO.3 and a downstream primer with a sequence as shown in SEQ ID NO.4; The primer pair for detecting AKT1 gene DNA mutation includes: an upstream primer with a sequence as shown in SEQ ID NO.1 and a downstream primer with a sequence as shown in SEQ ID NO.2; The primer pair for detecting the CTNNB1 gene DNA mutation is selected from at least one of the following pairs: an upstream primer with a sequence as shown in SEQ ID NO.33 and a downstream primer with a sequence as shown in SEQ ID NO.34; an upstream primer with a sequence as shown in SEQ ID NO.35 and a downstream primer with a sequence as shown in SEQ ID NO.36; an upstream primer with a sequence as shown in SEQ ID NO.37 and a downstream primer with a sequence as shown in SEQ ID NO.38; an upstream primer with a sequence as shown in SEQ ID NO.39 and a downstream primer with a sequence as shown in SEQ ID NO.40; The primer pair for detecting RAF1 gene DNA mutation 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.

4. The primer composition according to claim 3, characterized in that Also included are primer pairs for detecting RNA fusion variants in the ALK, BRAF, NTRK1, NTRK2, NTRK3, PPARG, RET, and THADA genes; The ALK gene RNA fusion variation includes the ALK gene and the EML4, KIF5B or TPM3 gene RNA fusion variation, and the primer pair includes: upstream primers with sequences as shown in SEQ ID NOs.551 to 556, SEQ ID NOs.561 to 566, and SEQ ID NOs.581 to 584, and downstream primers with sequences as shown in SEQ ID NOs.473 to 480; The BRAF gene RNA fusion variation is an RNA fusion variation of the BRAF gene and the AKAP9 gene, and the primer pair comprises: an upstream primer having a sequence as shown in SEQ ID NOs.485 to 490 and a downstream primer having a sequence as shown in SEQ ID NOs.481 to 484 and SEQ ID NOs.589 to 590; The NTRK1 gene RNA fusion variation is an RNA fusion variation of the NTRK1 gene and the TPM3 gene, and the primer pair comprises: an upstream primer having a sequence as shown in SEQ ID NOs.581 to 584 and a downstream primer having a sequence as shown in SEQ ID NOs.491 to 508; The NTRK2 gene RNA fusion variation is an RNA fusion variation of the NTRK2 gene and the BCR gene, and the primer pair includes: an upstream primer having a sequence as shown in SEQ ID NOs.591 to 592 and a downstream primer having a sequence as shown in SEQ ID NOs.509 to 518; The NTRK3 gene RNA fusion variation is an RNA fusion variation of the NTRK3 gene and the ETV6 gene, and the primer pair includes: upstream primers with sequences as shown in SEQ ID NOs.523 to 530 and SEQ ID NOs.557 to 560 and downstream primers with sequences as shown in SEQ ID NOs.519 to 522 and SEQ ID NOs.531 to 532; The PPARG gene RNA fusion variation is an RNA fusion variation of the PPARG gene and the PAX8 gene, and the primer pair comprises: an upstream primer having a sequence as shown in SEQ ID NOs.573 to 578 and a downstream primer having a sequence as shown in SEQ ID NOs.587 to 588; The RET gene RNA fusion variation includes the RNA fusion variation of the RET gene and the KIF5B, CCDC6, NCOA4 or PRKAR1A gene, and the primer pair includes: upstream primers with sequences as shown in SEQ ID NOs.561 to 566, SEQ ID NOs.545 to 550, SEQ ID NOs.567 to 572, and SEQ ID NOs.579 to 580, and downstream primers with sequences as shown in SEQ ID NOs.533 to 538; The THADA gene RNA fusion variation is an RNA fusion variation of the THADA gene and the IGF2BP3 gene, and the primer pair includes: an upstream primer whose sequence is shown in SEQ ID NOs.539 to 544 and a downstream primer whose sequence is shown in SEQ ID NOs.585 to 586.

5. The primer composition according to claim 4, characterized in that The primer composition includes primer pairs for detecting DNA mutations of 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 of ALK, BRAF, NTRK1, NTRK2, NTRK3, PPARG, RET, and THADA genes.

6. The primer composition according to claim 5, characterized in that At least 50% of the upstream primers in the primer composition have 5' ends connected to a nucleotide fragment with a sequence as shown in SEQ ID NO.593, and the corresponding downstream primers have 3' ends connected to a nucleotide fragment with a sequence as shown in SEQ ID NO.

594.

7. Use of the primer combination according to any one of claims 1 to 6 in the preparation of a product for detecting gene mutations associated with benign or malignant thyroid nodules.

8. The use according to claim 7, characterized in that The product is a test kit.

9. A kit for detecting gene mutations associated with benign and malignant thyroid nodules, characterized in that: The kit comprises the primer combination according to any one of claims 1 to 6.

10. A method for detecting gene mutations associated with benign and malignant thyroid nodules, characterized in that: The following steps are involved: Extract DNA and RNA from the sample to be tested, reverse transcribe the RNA to obtain cDNA; use the DNA and cDNA as templates, use the primer combination according to any one of claims 1 to 6 or the kit according to claim 9 to perform multiple PCR amplification, construct a library, and sequence.

11. A system for detecting gene mutations associated with benign and malignant thyroid nodules, characterized in that: include: Detection module: the detection module comprises 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 comprises the primer combination according to any one of claims 1 to 6 or the kit according to claim 9; Data analysis module: The data analysis module analyzes the sequencing results of the sequencing module, including filtering data below the quality control threshold, removing connectors, aligning sequences to corresponding positions of reference genome data, annotating gene mutations and statistically analyzing corresponding data.

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