WO2021139783A1 - 一次性检测肺癌多重基因突变的试剂盒 - Google Patents

一次性检测肺癌多重基因突变的试剂盒 Download PDF

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WO2021139783A1
WO2021139783A1 PCT/CN2021/070914 CN2021070914W WO2021139783A1 WO 2021139783 A1 WO2021139783 A1 WO 2021139783A1 CN 2021070914 W CN2021070914 W CN 2021070914W WO 2021139783 A1 WO2021139783 A1 WO 2021139783A1
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alk
detecting
lung cancer
seq
reagents
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French (fr)
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江风阁
黄蛤目
陈宁
李硕卿
蔡阿沙
郑伟伟
林小洪
宋庆涛
郑立谋
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厦门艾德生物医药科技股份有限公司
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Priority to JP2021554770A priority Critical patent/JP7204004B2/ja
Priority to EP21738110.2A priority patent/EP4089184A1/en
Publication of WO2021139783A1 publication Critical patent/WO2021139783A1/zh

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    • C12Q2600/16—Primer sets for multiplex assays
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Definitions

  • the invention belongs to the field of biotechnology, and more specifically, relates to a primer, a probe, a detection system and a kit for one-time detection of multiple gene mutations in lung cancer.
  • Lung cancer is one of the common malignant tumors that seriously endanger human health, and 80-85% of lung cancers are non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • the frequency of NTRK1, NTRK2, and NTRK3 gene fusion is about 3 ⁇ 7%, 1%, 1%, 0.12%, 0.02%, 0.08%, and the frequency of jumping gene mutation in MET exon 14 is about 1%.
  • a large number of clinical studies have shown that the driver gene mutation status is an important predictor of the efficacy of targeted drug therapy.
  • Patients with EGFR, HER2, KRAS, and BRAF gene mutations can benefit from the corresponding treatment with tyrosine kinase inhibitors; ALK, ROS1, RET Patients with NTRK1, NTRK2, and NTRK3 gene fusions can benefit from treatment with tyrosine kinase inhibitors; patients with MET exon 14 skipping gene mutations can benefit from treatment with MET inhibitors.
  • the NCCN Guidelines for Non-Small Cell Lung Cancer clearly pointed out that the gene mutation status needs to be tested before targeted therapy, and it is strongly recommended to conduct a wider range of effective gene status testing. Therefore, joint detection of multiple gene mutations in NSCLC patients can be Provide patients with more precise treatment.
  • the test results of this kit are for clinical reference only and should not be used as the only basis for individualized treatment of patients. Clinicians should comprehensively judge the test results based on the patient’s condition, drug indications, treatment response and other laboratory test indicators.
  • the invention provides a high-sensitivity and high-specificity primer, probe, kit and primer distribution method for detecting 11 lung cancer genes at one time.
  • the detection kit uses DNA and RNA samples to detect gene mutations and gene fusions respectively, with high detection specificity and sensitivity, good reproducibility of detection results, simple and quick operation, and is suitable for clinical EGFR, BRAF, HER2, KRAS, ALK, ROS1
  • the detection of 11 gene mutations/fusions of RET, NTRK1, NTRK2, NTRK3 and MET provides a fast and reliable detection method. This method can realize the PCR platform to detect 11 genes of lung cancer at one time, covering all the core gene targets of lung cancers that have been on the market and potentially marketed targeted drugs in the next 3-5 years. It will help clinical realization in the shortest time and with the least amount of use. Samples allow patients to benefit from precision medicine.
  • One of the objectives of the present invention is to provide a kit for detecting lung cancer gene mutations, which includes:
  • a reagent for detecting the A1 group site of lung cancer gene fusion includes EML4-ALK-1 to EML4-ALK-3, EML4-ALK-6 to EML4-ALK-14, EML4-ALK-17 To -EML4-ALK-21; KIF5B-ALK-1; KIF5B-ALK-2; KLC1-ALK; TFG-ALK; STRN-ALK-1; HIP1-ALK-1; HIP1-ALK-2; HIP1-ALK- 3; PRKAR1A-ALK-1; PRKAR1A-ALK-2; NBAS-ALK-1; TFG-ALK-2; PPM1B-ALK-1; EIF2AK3-ALK-1; BCL11A-ALK-1; BIRC6-ALK-1; CEBPZ-ALK-1; CLIP1-ALK-1; COL25A1-ALK-1; GCC2-ALK-1; GCC2-ALK-2; LMO7-ALK-21; KIF
  • a reagent for detecting lung cancer gene fusion site A5 includes ROS1-M1 to ROS1-M10;
  • a reagent for detecting B1 group sites of lung cancer mutation genes include E-19-M1 to E-19-M21; E-19-M24 to E-19-M27; E-20-M2;
  • a reagent for detecting lung cancer mutant gene B2 group sites include E-21-M1; E-18-M1; E-18-M2; E-18-M3;
  • the B3 group sites include E-20-M1; E-21-M2.
  • the five combinations of the present invention combine fusion genes (RNA part, A1 and A5, where A1 covers 46 fusions of lung cancer genes and ALK genes, and A5 covers 10 fusions of ROS1 genes) and mutant genes (DNA part B1, B2 and B3 cover 34 kinds of EGFR gene mutations) separately, so that DNA mutations and RNA fusion can be detected independently on the machine.
  • this design method is more user-friendly and avoids wasting reagents; the operation is also simpler and avoids incorrect additions or omissions caused by continuous switching of samples in the same reaction strip.
  • ALK and ROS1 fusion are tested separately in one tube, and EGFR is tested in three tubes, which saves precious clinical samples to the greatest extent. At the same time, it realizes the effective differentiation of different genes and different mutation types of the same gene, which better meets the clinical application.
  • Another object of the present invention is to provide a kit for detecting lung cancer gene mutations, including:
  • Reagents used to detect the following A1 group sites including EML4-ALK-1 to EML4-ALK-3, EML4-ALK-6 to EML4-ALK-14, EML4-ALK-17 to -EML4-ALK-21; KIF5B -ALK-1; KIF5B-ALK-2; KLC1-ALK; TFG-ALK; STRN-ALK-1; HIP1-ALK-1; HIP1-ALK-2; HIP1-ALK-3; PRKAR1A-ALK-1; PRKAR1A -ALK-2; NBAS-ALK-1; TFG-ALK-2; PPM1B-ALK-1; EIF2AK3-ALK-1; BCL11A–ALK-1; BIRC6-ALK-1; CEBPZ-ALK-1; CLIP1-ALK -1; COL25A1-ALK-1; GCC2-ALK-1; GCC2-ALK-2; LMO7-ALK-1; PICALM-AL
  • Reagents for detecting the following A2 sites including NTRK1-E9-M1; NTRK1-E10-M1; NTRK1-E10-M3; NTRK1-E10-M5 to NTRK1-E10-M9; NTRK1-E10-M12; NTRK1- E10-M14; NTRK1-E10-M15; NTRK1-E10-M17; NTRK1-E12-M1; NTRK1-E12-M3; NTRK1-E12-M4; NTRK1-E12-M11; NTRK1-E12-M12; NTRK1-E12- M14;
  • Reagents for detecting the following A3 group sites including NTRK2-E15-M1; NTRK2-E16-M1; NTRK2-E16-M3; NTRK2-E16-M7; NTRK2-E17-M2;
  • Reagents for detecting the following A4 sites including NTRK3-EX14-M1; NTRK3-EX14-M2; NTRK3-EX14-M3; NTRK3-EX14-M4; NTRK3-EX14-M7; NTRK3-EX15-M1; NTRK3- EX15-M2; NTRK3-EX15-M3;
  • Reagents for detecting the following A5 sites including ROS1-M1 to ROS1-M10;
  • Reagents for detecting the following A6 sites including ROS1-M11; ROS1-M12; ROS1-M13; ROS1-EX35-M1 to ROS1-EX35-M4; ROS1-EX35-M6; ROS1-EX35-M8 to ROS1- EX35-M11; ROS1-EX35-M13;
  • Reagents used to detect the following A7 sites including MET-M1;
  • Reagents used to detect the following A8 sites including RET-M2; RET-M5; RET-M15; RET-M16; RET-M17; RET-M19; LRET-M22; LRET-M32; LRET-M40; LRET- M41; LRET-M42; LRET-M44; LRET-M45; LRET-M49; LRET-M55; LRET-M57; LRET-M58; LRET-M59;
  • Reagents for detecting the following B1 sites including E-19-M1 to E-19-M21; E-19-M24 to E-19-M27; E-20-M2;
  • Reagents for detecting the following B2 sites including E-21-M1; E-18-M1; E-18-M2; E-18-M3;
  • Reagents used to detect the following B3 sites including E-20-M1; E-21-M2;
  • Reagents for detecting the following B4 sites including E-20-M4; E-20-M5; E-20-M8; E-20-M9; E-20-M13; E-20-M14; E- 20-M20; E-20-M21; E-20-M23; E-20-M24; E-20-M28 to E-20-M31; E-20-M36 to E-20-M41; E-20- M44; E-20-M48; E-20-M50 to E-20-M53; BRAF-M1;
  • Reagents for detecting the following B5 sites including E-20-M3; E-20-M10; E-20-M12; E-20-M15 to E-20-M19; E-20-M22; E- 20-M25 to E-20-M27; E-20-M32 to E-20-M35; E-20-M42; E-20-M43; E-20-M45 to E-20-M47; E-20- M49; E-20-M55 to E-20-M57; KRAS-M6;
  • Reagents for detecting the following B6 group sites including HER2-M1 to HER2-M3; HER2-M8; HER2-M9; HER2-M15 to HER2-M26; KRAS-M2; KRAS-M3; KRAS-M5; KRAS- M14;
  • Reagents for detecting the following B7 group sites including HER2-M4; HER2-M6; HER2-M7; HER2-M10; KRAS-M1; KRAS-M4.
  • the 15 reagent combinations of the kit of the present invention cover seven fusion genes of ALK, ROS1, RET, NTRK1, NTRK2, NTRK3, and MET and four mutant genes of EGFR, KRAS, BRAF, and HER2, with a total of 119 gene fusions and 112 genes Mutations, specifically including, 83 mutations in EGFR gene, 7 mutations in KRAS gene, 1 mutation in BRAF gene, 21 mutations in HER2 gene, 46 fusions in ALK gene, 23 fusions in ROS1 gene, 18 fusions in RET gene, 18 fusions in NTRK1 gene Two fusions, five fusions of NTRK2 gene, eight fusions of NTRK3 gene and one jumping mutation of MET gene (see Table 5 and Table 6 for details).
  • the approved multi-gene combined detection kits for lung cancer or authorized multi-gene combined detection kits on the market use a PCR 8 strip or 12 strip, and mutation and fusion detection are placed in the same reaction strip.
  • PCR method PCR 8 strip or 12 strip
  • mutation and fusion detection are placed in the same reaction strip.
  • the 15 combinations of the kit of the present invention plus an external control can be divided into 2 8-link PCR reaction strips, that is, each reagent in the kit can contain 2 8-link PCR reaction strips, and the fusion gene (RNA part ) And mutant gene (DNA part), respectively, a separate 8-link PCR reaction strip, so that DNA mutation and RNA fusion can be detected on the machine independently.
  • this design method is more user-friendly and avoids wastage of reagents; the operation is also easier to avoid incorrect additions and omissions caused by continuous switching of samples in the same reaction strip; in addition, 8
  • the consumables of the reaction strip are more applicable, the required equipment is simpler, and the clinical promotion and use are easier.
  • test site When the test site is combined and managed by the kit, it is comprehensively considered from the clinical significance of the gene, the needs of the pharmaceutical manufacturer, the ease of operation, the interaction between different genes, the methodological feasibility, and the best performance indicators for each gene locus. . For example, it is necessary to consider whether different genes, different exons of the same gene, and different codons have different clinical significance, combine genes/sites with the same clinical significance as much as possible, and distinguish sites with different clinical significance independently.
  • patients with EGFR T790M mutations are significantly inferior to other EGFR sensitive mutations in the objective response rate and disease progression-free survival time of the first-generation EGFR-TKI, but are sensitive to the third-generation EGFR inhibitor osimertinib; another example is the KRAS gene mutation. It is an unfavorable factor that affects the efficacy of TKI-type targeted drugs. As long as there are KRAS gene mutations, common targeted drugs targeting EGFR, ALK, ROS1 and other genes cannot be used. In addition, lung cancer patients with KRAS gene mutations often do not have the effect of chemotherapy drugs. Good.
  • First-line drugs such as pemetrexed and paclitaxel combined with platinum-based chemotherapy are less effective than patients without KRAS mutations.
  • multiple targeted or immunotherapeutic drugs have shown excellent results in patients with KRAS mutations.
  • AMG 510 is the first KRAS G12C inhibitor to reach the clinical stage in recent years.
  • the results of the AMG 510 Phase I study show that the total ORR (objective response rate) of AMG 510 for NSCLC (non-small cell lung cancer) patients with KRAS mutations is 48% and DCR (Disease Control Rate) 96%.
  • Another object of the present invention is to provide a composition for the one-time detection of multiple lung cancer gene mutations, including primers for EGFR, KRAS, BRAF, HER2, ALK, ROS1, RET, NTRK1, NTRK2, NTRK3, MET and other mutant genes ⁇ Probes, the above primers and probes are divided into two sets of PCR 8-tubes, as shown in Table 1:
  • the present invention also provides a reaction system of the aforementioned primers and probes, including:
  • Reverse transcription reaction system purified water 5 ⁇ 25 ⁇ L, 5 ⁇ reverse transcription buffer 5 ⁇ 15 ⁇ L, each primer 20 ⁇ 150 ⁇ mol, reverse transcriptase 100 ⁇ 200U, total volume is 20 ⁇ 30 ⁇ L;
  • (2) PCR reaction system purified water 15 ⁇ 45 ⁇ L, 10 ⁇ PCR buffer 5 ⁇ 15 ⁇ L, MgCl 2 1 ⁇ 10mmol, each pair of primers 1 ⁇ 20pmol, each pair of probes 1 ⁇ 20pmol, dNTPs10 ⁇ 100pmol, Taq enzyme 1 ⁇ 10U, the total volume is 30 ⁇ 50 ⁇ L.
  • kits which includes the sequence of SEQ ID NO: 1 to 281.
  • the kit includes two PCR 8-strip tubes, and two PCR 8-strip tubes respectively detect lung cancer RNA gene fusion and lung cancer DNA gene mutations.
  • the kit contains specific primers and probes for genes such as EGFR, KRAS, BRAF, HER2, ALK, ROS1, RET, NTRK1, NTRK2, NTRK3, MET, etc. It also includes PCR buffer, UNG enzyme, etc.
  • the PCR 8-tube detection reagents for detecting lung cancer RNA gene fusion include: 1 tube each of ALK, NTRK1, NTRK2, and NTRK3 fusion gene detection reagents, 2 tubes of ROS1 fusion gene detection reagents, and MET 14 exon skipping gene mutation detection 1 tube of reagent, 1 tube of RET fusion gene detection reagent.
  • Tubes 1 to 4 of the eight-pipe tube consist of ALK, NTRK1, NTRK2 and NTRK3 fusion gene detection reagents respectively
  • tubes 5 and 6 are composed of ROS1 fusion gene detection reagents
  • tube 7 is MET 14 exon skip mutation detection Reagent composition, No.
  • tube is composed of RET fusion gene detection reagent and internal control detection reagent.
  • the fusion gene detection reagents in tubes 1-8 are indicated by the FAM signal, and the internal control detection reagents in tubes 1-8 are used to monitor the quality of sample RNA and the addition status, and are indicated by the VIC signal.
  • the PCR 8-tube detection reagents for detecting lung cancer DNA gene mutations include: 3 tubes of EGFR mutation gene detection reagents, 1 tube of EGFR/BRAF mutation gene detection reagents, 1 tube of EGFR/KRAS mutation gene detection reagents, and HER2/KRAS mutation gene detection. 2 tubes of reagents, 1 tube of external control detection reagents.
  • the FAM signal of tubes 1 to 5 and the ROX signal of tubes 1 to 3 in the octagonal tube respectively indicate different mutation sites of the EGFR gene.
  • the ROX signal of tube 4 indicates the V600E mutation of the BRAF gene, and tubes 6 to 7
  • the FAM signal indicates different mutation sites of the HER2 gene
  • the ROX signals of tubes 5-7 indicate different mutation sites of the KRAS gene
  • the internal control detection reagents of tubes 1-7 are used to detect the addition of samples, indicated by the VIC signal
  • Tube 8 is composed of detection reagents that amplify conserved segments of the human genome. It is used to monitor the quality of sample DNA and is indicated by FAM and ROX signals.
  • Another object of the present invention is to provide a method of using the kit, the steps of which include:
  • the test sample includes fresh pathological tissue, paraffin-embedded tissue or paraffin sectioned tissue;
  • the present invention adopts specific primer and probe technology, three-color fluorescence channel detection mode, and establishes a real-time PCR system, which can simultaneously detect ALK, ROS1, RET, NTRK1, NTRK2, NTRK3, MET seven fusion genes and EGFR, KRAS , BRAF, HER2 four mutant genes, a total of 119 gene fusions and 112 gene mutations, specifically including 83 mutations in EGFR gene, 7 mutations in KRAS gene, 1 mutation in BRAF gene, 21 mutations in HER2 gene and 46 in ALK gene Two fusions, 23 fusions of ROS1 gene, 18 fusions of RET gene, 18 fusions of NTRK1 gene, 5 fusions of NTRK2 gene, 8 fusions of NTRK3 gene, and 1 jump mutation of MET gene (see Table 5 and Table 6 for details).
  • the present invention divides 281 sequences (including primers and probes) into groups based on the detection difficulty of multiple mutations/fusion sites, the mutual influence between primers, competition and other comprehensive factors.
  • double or triple PCR is used to combine multiple mutation types and distinguish them with different fluorescently labeled probes, which can detect multiple mutations in one tube. Types are convenient for customers to operate, while saving testing costs and testing samples; (2) When merging mutation types, in addition to considering the difficulty of technical merging, it also takes into account the needs of different drugs.
  • the kit can detect 231 mutations/fusion sites at one time , And the operation method/amplification procedure of each reaction tube is basically the same, easy to operate, saving time, and convenient for clinical application;
  • Each reaction system also contains an internal control detection system, and the internal control of the fusion reaction tube is used for quality control RNA samples
  • the internal control of the mutation reaction tube is used to control the quality and operation of the DNA sample and whether the equipment is abnormal.
  • the external control is used to judge the quality of the sample DNA and participate in the interpretation of the results.
  • the detection sites covered by the kit provided by the present invention account for more than 95% of all mutation types of each gene.
  • the total mutation rate in non-small cell lung cancer patients is as high as 70%, which is currently the most detected mutation genes and mutation types.
  • the most complete fluorescent PCR product provides a comprehensive reference for clinicians to choose tumor-targeted drug treatment for patients with lung cancer.
  • the kit provided by the present invention is simple and quick to operate, has high sensitivity, good specificity, objective result interpretation, etc., and becomes the most popular clinically popular first-line detection method.
  • Fig. 1 is a sensitivity diagram of ALK fusion genes—EML4 exon 13, ALK exon 20 in Example 1.
  • Figure 2 is a sensitivity diagram of the ROS1 fusion gene-CD74 exon 6, ROS1 exon 32 in Example 1.
  • Fig. 3 is a sensitivity graph of the RET fusion gene-KIF5B exon 16, ROS1 exon 12 in Example 1.
  • Fig. 4 is a sensitivity graph of NTRK1 fusion gene-CD74 exon8 and NTRK1 exon10 in Example 1.
  • Fig. 5 is a sensitivity graph of NTRK2 fusion genes-TRIM24 exon12 and NTRK2 exon15 in Example 1.
  • Fig. 6 is a sensitivity graph of NTRK3 fusion gene-ETV6 exon5, NTRK3 exon15 in Example 1.
  • Figure 7 is a sensitivity graph of MET skipping mutation-MET exon14 skipping in Example 1.
  • Figure 8 is a sensitivity graph of EGFR gene mutation -19del in Example 1.
  • Figure 9 is a sensitivity graph of EGFR gene mutation-L858R in Example 1.
  • Figure 10 is a sensitivity graph of the EGFR gene mutation-T790M in Example 1.
  • Figure 11 is a sensitivity graph of KRAS gene mutation-G12D in Example 1.
  • Figure 12 is a sensitivity graph of KRAS gene mutation-G12C in Example 1.
  • Figure 13 is a sensitivity graph of BRAF gene mutation-V600E in Example 1.
  • Figure 14 is a sensitivity graph of the HER2 gene mutation-V769_D770insASV in Example 1.
  • Figure 15 is a sensitivity graph of the HER2 gene mutation-A775_G776insYVMA in Example 1.
  • 16 is a PCR diagram of the ALK fusion gene detection result of the clinically positive sample in Example 2.
  • Figure 17 is a PCR diagram of the ROS1 fusion gene detection result of the clinically positive sample in Example 2.
  • Example 18 is a PCR diagram of the detection result of the RET fusion gene of the clinical positive sample in Example 2.
  • FIG. 19 is a PCR diagram of the detection result of NTRK3 fusion gene in a clinically positive sample in Example 2.
  • FIG. 19 is a PCR diagram of the detection result of NTRK3 fusion gene in a clinically positive sample in Example 2.
  • FIG. 20 is a PCR diagram of the detection result of MET skip mutation in a clinically positive sample in Example 2.
  • 21 is a PCR diagram of the detection result of the EGFR19del gene mutation in the clinically positive sample in Example 2.
  • Figure 22 is a PCR diagram of the detection results of the EGFR L858R gene mutation in the clinically positive sample in Example 2.
  • FIG. 23 is a PCR diagram of the detection result of the EGFR T790M gene mutation in the clinically positive sample in Example 2.
  • Example 24 is a PCR diagram of the KRAS gene mutation detection result of the clinically positive sample in Example 2.
  • Example 25 is a PCR diagram of the KRAS G12C gene mutation detection result of the clinical positive sample in Example 2.
  • 26 is a PCR diagram of the BRAF gene mutation detection result of the clinically positive sample in Example 2.
  • FIG. 27 is a PCR diagram of the detection result of HER2 gene mutation in a clinically positive sample in Example 2.
  • ALK fusion genes EML4 exon 13, ALK exon 20, ROS1 fusion gene CD74 exon 6, ROS1 exon 32, RET fusion gene KIF5B exon 16, ROS1 exon 12, NTRK1 fusion gene CD74 exon8, NTRK1 exon10, NTRK2 fusion gene TRIM24 exon12, NTRK2 exon15, NTRK3 fusion gene ETV6 exon5, NTRK3 exon15, MET jumping mutation gene MET exon14 skipping, EGFR gene mutation gene 19del, L858R, T790M, KRAS mutation gene G12D, G12C, BRAF gene mutation gene V600E, HER2 gene mutation gene V769_D770insASV and A775_G776insYVMA are taken as examples to illustrate the fluorescent PCR detection of the above-mentioned lung cancer genes of the present invention.
  • the experiment uses T7RNA containing the above-mentioned fusion gene and each mutant plasmid template, and the fluorescent PCR detection includes the following steps:
  • the plasmid was extracted with Tiangen Biochemical Plasmid Small Extraction Kit (Cat. No.: DP103), and the plasmid was transcribed with TranscriptAid T7 High Yield Transcription Kit (Cat. No.: K0441) to obtain T7 RNA.
  • the specific operation steps followed the kit instructions.
  • the OD 260 /OD 280 of DNA and RNA should be between 1.7 and 2.1; the concentration of RNA should be between 10 and 100 ng/ ⁇ L.
  • the template obtained above is used as a template for real-time fluorescent PCR amplification, and PCR amplification is carried out according to the following amplification system:
  • Reverse transcription reaction system purified water 5 ⁇ 25 ⁇ L, 5 ⁇ reverse transcription buffer 5 ⁇ 15 ⁇ L, each primer 20 ⁇ 150 ⁇ mol, reverse transcriptase 100 ⁇ 200U, total volume is 20 ⁇ 30 ⁇ L;
  • PCR reaction system purified water 15 ⁇ 45 ⁇ L, 10 ⁇ PCR buffer solution 5 ⁇ 15 ⁇ L, MgCl 2 1 ⁇ 10mmol, each pair of primers 1 ⁇ 20pmol, each pair of probes 1 ⁇ 20pmol, dNTPs10 ⁇ 100pmol, Taq enzyme 1 ⁇ 10U, The total volume is 30-50 ⁇ L.
  • Reverse transcription reaction conditions 42°C60min, 95°C5min, 1 cycle;
  • the real-time PCR reaction conditions are:
  • the first stage 42°C5min, 95°C5min, 1 cycle;
  • the second stage 95°C for 25s, 64°C for 20s, 72°C for 20s, 10 cycles;
  • the third stage 93°C for 25s, 60°C for 35s, 72°C for 20s, 36 cycles;
  • Signal collection Collect FAM/VIC/ROX signals at 60°C in the third stage, perform real-time PCR, and save files.
  • Negative control LET PCR 8-strip-gene fusion-1 ⁇ 8FAM signal and LET PCR 8-strip-gene mutation-1 ⁇ 7FAM and ROX signal should have no amplification curve rise, if any of the above signals have an amplification curve rise , The result of this experiment is invalid, it is recommended to re-test.
  • RNA may be fragmented or RNA degraded, or the sample may contain inhibitors or omissions. It is recommended to retest or re-extract RNA before testing.
  • the positive sample of tube 5 may have cross-reaction amplification in tube 6. Therefore, when both tubes 5 and 6 have positive amplification curves, it is determined that tube 5 is a true positive amplification signal, and tube 6 is Cross-reaction amplification signal.
  • any one of tubes 1 to 7 is ⁇ 36, it indicates that the DNA may be fragmented or DNA degraded, or the sample may contain inhibitors or omissions. It is recommended to re-test or re-extract the DNA before testing.
  • the Ct value of the FAM and ROX signal of tube 8 is greater than 25, it means that the added DNA may be fragmented or degraded, or there may be inhibitors or omissions. If the mutation signal rises and falls in the positive A zone, the experimental result is still It is credible, otherwise it is recommended to increase the amount of DNA sample or re-extract DNA before testing
  • the ⁇ Ct value of the reaction tube is calculated. If the ⁇ Ct value of the reaction tube is less than the corresponding ⁇ Ct Cut-off value, the sample is also positive for the mutation corresponding to the reaction tube, that is, positive zone B; otherwise, it is negative or lower than the lower detection limit of the kit.
  • ⁇ Ct value sudden change FAM (ROX) Ct value-external control FAM (ROX) Ct value.
  • the mutation FAM (ROX) Ct value refers to the Ct value corresponding to the sample mutation signal No. 1-7 (FAM or ROX signal);
  • the external control Ct value refers to the sample corresponding to the external control signal No. 8 (FAM or ROX signal) Ct value.
  • Certain EGFR, HER2, KRAS positive samples may cause cross signals between individual mutation reaction tubes.
  • the sample has a positive result in 2 or more reaction tubes (interpreted in accordance with Table 10)
  • first determine that the mutation reaction tube with the smallest Ct value is a true positive and then calculate the ⁇ Ct value of each reaction tube (mutation Ct value-external Control the Ct value), and determine whether the other reaction tubes are cross-signals according to the cross-signal thresholds listed in Tables 11-13.
  • the mutation reaction tube can be judged as negative.
  • a sample may contain 2 or more fusion or mutation types at the same time.
  • the detection result shows that the detection system of the present invention can accurately detect 11 lung cancer genes at one time, and the detection sensitivity can reach 1% of gene mutation DNA and 25 copies/ ⁇ L of fusion gene RNA.
  • DNA and RNA are extracted using nucleic acid extraction reagents from Xiamen Aide Biomedical Technology Co., Ltd. (model: FFPE DNA/RNA, medical device record number: Minxia Xie Bei No. 20150082, article number : 8.0223601X036G).
  • concentration and purity of DNA and RNA are detected by a micro-ultraviolet spectrophotometer.
  • concentration of RNA should be greater than 10ng/ ⁇ L
  • the concentration of DNA should be greater than 2ng/ ⁇ L
  • the OD 260 /OD 280 of DNA and RNA should be between 1.7 and 2.1.
  • the DNA and RNA extracted above were used as amplification templates for the detection of 11 lung cancer genes.
  • Reverse transcription reaction system purified water 5 ⁇ 25 ⁇ L, 5 ⁇ reverse transcription buffer 5 ⁇ 15 ⁇ L, each primer 20 ⁇ 150 ⁇ mol, reverse transcriptase 100 ⁇ 200U, total volume is 20 ⁇ 30 ⁇ L;
  • PCR reaction system purified water 15 ⁇ 45 ⁇ L, 10 ⁇ PCR buffer 5 ⁇ 15 ⁇ L, MgCl2 1 ⁇ 10mmol, each pair of primers 1 ⁇ 20pmol, each pair of probes 1 ⁇ 20pmol, dNTPs10 ⁇ 100pmol, Taq enzyme 1 ⁇ 10U, total The volume is 30-50 ⁇ L.
  • Reverse transcription reaction conditions 42°C60min, 95°C5min, 1 cycle;
  • S-cDNA1 and S-cDNA2 are obtained, mixed with enzymes, and added to the detection tube LET PCR 8 strips-gene fusion -1 ⁇ 4 and 5 ⁇ 8; DNA samples are added to the detection tube LET PCR 8 strips-gene mutation From -1 to 8, perform PCR amplification.
  • the real-time PCR reaction conditions are:
  • the first stage 42°C5min, 95°C5min, 1 cycle;
  • the second stage 95°C for 25s, 64°C for 20s, 72°C for 20s, 10 cycles;
  • the third stage 93°C for 25s, 60°C for 35s, 72°C for 20s, 36 cycles;
  • Signal collection Collect FAM/VIC/ROX signals at 60°C in the third stage, perform real-time PCR, and save files.
  • the detection result is judged according to the Ct value displayed by the fluorescent PCR amplification instrument (refer to Example 1 for the judgment method).
  • NGS detection Qualified DNA samples were extracted, fragmented, and then repaired and connected with adapters. The target area was captured using the Aide pan-tumor 116 gene panel capture probe. The captured library qualified for quality control was sequenced using the Illumina Novaseq 6000 sequencing platform. , And used the Aide pan-cancer species 116 gene mutation joint detection data analysis system (ADXPAN116-tMut_v0.2.0) to analyze the sequencing data, and obtained 116 genes (including EGFR, KRAS, BRAF, HER2, ALK, ROS1, RET, NTRK1) , NTRK2, NTRK3, MET, etc.).
  • ADXPAN116-tMut_v0.2.0 Aide pan-cancer species 116 gene mutation joint detection data analysis system
  • the total coincidence rate of the fluorescent PCR method and NGS results is as high as 99.42%, and the sensitivity and selective detection ability of fluorescent PCR are equivalent to those of NGS.
  • the PCR method is convenient and quick to detect 11 genes, takes a short time, saves samples, and is more conducive to clinical application.
  • the invention discloses a kit for detecting multiple gene mutations of lung cancer, which includes primers and probes for detecting mutations of lung cancer EGFR, KRAS, BRAF, HER2, ALK, ROS1, RET, NTRK1, NTRK2, NTRK3, MET genes And its distribution method.
  • the present invention adopts a PCR 8-tube design, every two PCR 8-tubes detects 11 gene mutations/fusion states of a sample, and one PCR 8-tube is equipped with corresponding fusion detection reagents and internal control reagents; the other PCR 8-tube is equipped with corresponding fusion detection reagents and internal control reagents. Corresponding mutation detection reagents are installed in the tube.
  • the invention uses the fluorescent PCR method to detect 119 gene fusions and 112 gene mutation types of lung cancer at one time, greatly shortens the detection time, is simple and convenient to operate, accurate results, and has industrial practicability.

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Abstract

本发明公开了一种一次性检测肺癌多重基因突变的组合物及其应用，其中包括用于检测肺癌EGFR、KRAS、BRAF、HER2、ALK、ROS1、RET、NTRK1、NTRK2、NTRK3、MET基因突变的引物、探针及其分配方式。本发明采用PCR 8联管设计，每两个PCR 8联管检测一个样品的11种基因突变/融合状态，其中一个PCR 8联管内装有相应的融合检测试剂和内控试剂；另一个PCR 8联管管内装有相应的突变检测试剂。

Description

一次性检测肺癌多重基因突变的试剂盒 技术领域
本发明属于生物技术领域，更具体的，涉及一种一次性检测肺癌多重基因突变的引物、探针、检测体系和试剂盒。
背景技术
肺癌是严重危害人类健康的常见恶性肿瘤之一，其中80～85％的肺癌为非小细胞肺癌(NSCLC)。NSCLC患者中存在多种基因突变类型，其中EGFR、HER2、KRAS、BRAF基因突变频率分别约为10～50％、1～4％、5～25％、1～2％，ALK、ROS1、RET、NTRK1、NTRK2、NTRK3基因融合发生频率分别约为3～7％、1％、1％、0.12％、0.02％、0.08％，MET外显子14跳跃基因突变发生频率约为1％。大量临床研究表明，驱动基因突变状态是靶向药物治疗的重要疗效预测因子，EGFR、HER2、KRAS、BRAF基因突变患者可以从相应的酪氨酸激酶抑制剂治疗中获益；ALK、ROS1、RET、NTRK1、NTRK2、NTRK3基因融合患者可以从酪氨酸激酶抑制剂治疗中获益；MET外显子14跳跃基因突变患者可以从MET抑制剂治疗中获益。《非小细胞肺癌NCCN指南》明确指出，在进行靶向治疗前需要对基因突变状态进行检测，并强烈建议进行更广泛的有效基因的状态检测，因此，对NSCLC患者的多基因突变联合检测可为患者提供更精准的治疗。本试剂盒检测结果仅供临床参考，不应作为患者个体化治疗的唯一依据，临床医生应结合患者病情、药物适应症、治疗反应及其它实验室检测指标等因素对检测结果进行综合判断。
这些基因突变或融合状态与靶向药物的疗效相关，多基因的联合检测可进一步提高预测预后的准确性。然而，目前大多数试剂盒每次只能检测一种或几种基因突变，检测通量低，耗时长，对样本需求量大。虽然目前市面上用于科研用途的NGS Panel也有可能一次性检测上述11种基因，但NGS价格昂贵、操作繁琐、数据分析复杂、样本用量多等因素也制约了NGS方法的推广应用。目前临床上尚无能够同时、一次性完成对此11个热点基因进行检测的PCR试剂盒。本发明提供一种高灵敏度和高特异性的用于一次性检测11种肺癌基因的引物、探针、试剂盒及引物分配方式。该检测试剂盒利用DNA和RNA样本，分别进行基因突变和基因融合的检测，检测特异性和灵敏度高，检测结果重复性好，操作简便快捷，为临床EGFR、BRAF、HER2、KRAS、ALK、ROS1、RET、NTRK1、NTRK2、NTRK3和MET 11种基因突变/融合 的检测提供了一种快速可靠的检测方法。该方法可以实现PCR平台一次性检测肺癌的11种基因，涵盖了肺癌已上市及未来3-5年潜在上市靶向药物所有的核心基因靶点，将助力临床实现在最短的时间、用最少的样本让患者从精准医疗中获益。
发明内容
本发明的目的之一，在于提供一种检测肺癌基因突变的试剂盒，其包括：
用于检测肺癌基因融合的A1组位点的试剂，所述A1组位点包括EML4-ALK-1至EML4-ALK-3、EML4-ALK-6至EML4-ALK-14、EML4-ALK-17至-EML4-ALK-21；KIF5B-ALK-1；KIF5B-ALK-2；KLC1-ALK；TFG-ALK；STRN-ALK-1；HIP1-ALK-1；HIP1-ALK-2；HIP1-ALK-3；PRKAR1A-ALK-1；PRKAR1A-ALK-2；NBAS-ALK-1；TFG-ALK-2；PPM1B-ALK-1；EIF2AK3-ALK-1；BCL11A–ALK-1；BIRC6-ALK-1；CEBPZ-ALK-1；CLIP1-ALK-1；COL25A1-ALK-1；GCC2-ALK-1；GCC2-ALK-2；LMO7-ALK-1；PICALM-ALK-1；PHACTR1-ALK-1；TPR-ALK-1；MPRIP-ALK-1；TNIP2-ALK-1；DCTN1-ALK-1；SQSTM1-ALK-1；
用于检测肺癌基因融合A5组位点的试剂，所述第A5组位点包括ROS1-M1至ROS1-M10；
用于检测肺癌突变基因B1组位点的试剂，所述B1组位点包括E-19-M1至E-19-M21；E-19-M24至E-19-M27；E-20-M2；
用于检测肺癌突变基因B2组位点的试剂，所述B2组位点包括E-21-M1；E-18-M1；E-18-M2；E-18-M3；
用于检测肺癌突变基因B3组位点的试剂：所述B3组位点包括E-20-M1；E-21-M2。
本发明的这5种组合，是将融合基因(RNA部分，A1和A5，其中A1涵盖了肺癌基因的ALK基因46种融合，A5涵盖了ROS1基因10种融合)和突变基因(DNA部分B1、B2、B3涵盖了EGFR基因34种突变)分开，使DNA突变和RNA融合能够独立上机检测。对于仅需要检测DNA突变或RNA融合的样本，该设计方式更加人性化，避免反应试剂的浪费；操作也更加简便，避免同一个反应条中因样本不断切 换导致的错加漏加。
ALK和ROS1融合分别独立1管检测，EGFR分3管进行检测，最大程度地节约了珍贵的临床样本，同时实现了不同基因及同一基因不同突变类型的有效区分，更好地满足了临床应用。
进一步，本发明的另一目的，在于提供一种检测肺癌基因突变的试剂盒，包括：
包括15个组合，其中：
用于检测如下A1组位点的试剂：包括EML4-ALK-1至EML4-ALK-3、EML4-ALK-6至EML4-ALK-14、EML4-ALK-17至-EML4-ALK-21；KIF5B-ALK-1；KIF5B-ALK-2；KLC1-ALK；TFG-ALK；STRN-ALK-1；HIP1-ALK-1；HIP1-ALK-2；HIP1-ALK-3；PRKAR1A-ALK-1；PRKAR1A-ALK-2；NBAS-ALK-1；TFG-ALK-2；PPM1B-ALK-1；EIF2AK3-ALK-1；BCL11A–ALK-1；BIRC6-ALK-1；CEBPZ-ALK-1；CLIP1-ALK-1；COL25A1-ALK-1；GCC2-ALK-1；GCC2-ALK-2；LMO7-ALK-1；PICALM-ALK-1；PHACTR1-ALK-1；TPR-ALK-1；MPRIP-ALK-1；TNIP2-ALK-1；DCTN1-ALK-1；SQSTM1-ALK-1；
用于检测如下A2组位点的试剂：包括NTRK1-E9-M1；NTRK1-E10-M1；NTRK1-E10-M3；NTRK1-E10-M5至NTRK1-E10-M9；NTRK1-E10-M12；NTRK1-E10-M14；NTRK1-E10-M15；NTRK1-E10-M17；NTRK1-E12-M1；NTRK1-E12-M3；NTRK1-E12-M4；NTRK1-E12-M11；NTRK1-E12-M12；NTRK1-E12-M14；
用于检测如下A3组位点的试剂：包括NTRK2-E15-M1；NTRK2-E16-M1；NTRK2-E16-M3；NTRK2-E16-M7；NTRK2-E17-M2；
用于检测如下A4组位点的试剂：包括NTRK3-EX14-M1；NTRK3-EX14-M2；NTRK3-EX14-M3；NTRK3-EX14-M4；NTRK3-EX14-M7；NTRK3-EX15-M1；NTRK3-EX15-M2；NTRK3-EX15-M3；
用于检测如下A5组位点的试剂：包括ROS1-M1至ROS1-M10；
用于检测如下A6组位点的试剂：包括ROS1-M11；ROS1-M12；ROS1-M13；ROS1-EX35-M1至ROS1-EX35-M4；ROS1-EX35-M6；ROS1-EX35-M8至ROS1-EX35-M11；ROS1-EX35-M13；
用于检测如下A7组位点的试剂：包括MET-M1；
用于检测如下A8组位点的试剂：包括RET–M2；RET–M5；RET-M15；RET–M16；RET–M17；RET–M19；LRET-M22；LRET-M32；LRET-M40；LRET-M41；LRET-M42；LRET-M44；LRET-M45；LRET-M49；LRET-M55；LRET-M57；LRET-M58；LRET-M59；
用于检测如下B1组位点的试剂：包括E-19-M1至E-19-M21；E-19-M24至E-19-M27；E-20-M2；
用于检测如下B2组位点的试剂：包括E-21-M1；E-18-M1；E-18-M2；E-18-M3；
用于检测如下B3组位点的试剂：包括E-20-M1；E-21-M2；
用于检测如下B4组位点的试剂：包括E-20-M4；E-20-M5；E-20-M8；E-20-M9；E-20-M13；E-20-M14；E-20-M20；E-20-M21；E-20-M23；E-20-M24；E-20-M28至E-20-M31；E-20-M36至E-20-M41；E-20-M44；E-20-M48；E-20-M50至E-20-M53；BRAF-M1；
用于检测如下B5组位点的试剂：包括E-20-M3；E-20-M10；E-20-M12；E-20-M15至E-20-M19；E-20-M22；E-20-M25至E-20-M27；E-20-M32至E-20-M35；E-20-M42；E-20-M43；E-20-M45至E-20-M47；E-20-M49；E-20-M55至E-20-M57；KRAS-M6；
用于检测如下B6组位点的试剂：包括HER2-M1至HER2-M3；HER2-M8；HER2-M9；HER2-M15至HER2-M26；KRAS-M2；KRAS-M3；KRAS-M5；KRAS-M14；
用于检测如下B7组位点的试剂：包括HER2-M4；HER2-M6；HER2-M7；HER2-M10；KRAS-M1；KRAS-M4。
本发明试剂盒的15种试剂组合，涵盖ALK、ROS1、RET、NTRK1、NTRK2、NTRK3、MET七种融合基因和EGFR、KRAS、BRAF、HER2四种突变基因，共有119种基因融合和112种基因突变，具体包括，EGFR基因83种突变、KRAS基因7种突变、BRAF基因1种突变、HER2基因21种突变以及ALK基因46种融合、ROS1基因23种融合、RET基因18种融合、NTRK1基因18种融合、NTRK2基因5种融合、NTRK3基因8种融合和MET基因1种跳跃突变(详见表5、表6)。
目前市面上获批的肺癌多基因联合检测试剂盒(PCR法)或授权的多基因联合检测试剂盒都是采用一个PCR 8联条或12联条，突变和融合检测分别放在同一个反应条的不同位置，上机检测时，需要将整个反应条放入PCR仪器中进行扩增。
而本发明试剂盒的15种组合再加一个外控，可以分在2个8联PCR反应条，也即可以在试剂盒每人份试剂包含2个8联PCR反应条，融合基因(RNA部分)和突变基因(DNA部分)分别单独一个8联PCR反应条，使DNA突变和RNA融合能够独立上机检测。对于仅需要检测DNA突变或RNA融合的样本，该设计方式更加人性化，避免反应试剂的浪费；操作也更加简便，避免同一个反应条中因样本不断切换导致的错加漏加；此外，8联反应条的耗材适用性更广，需要的仪器设备更简单，临床推广使用更容易。
试剂盒在检测位点组合并管时，从基因临床意义、药厂需求、操作简便程度、不同基因间相互作用、方法学可行性、每个基因位点最佳性能指标等几个方向综合考虑。例如，需要考虑不同基因、同一基因不同外显子、不同密码子是否具有不同的临床意义，将临床意义相同的基因/位点尽量进行合并，将临床意义不同的位点进行独立区分。比如携带有EGFR T790M突变的患者使用一代EGFR-TKI的客观反应率和无疾病进展生存期显著劣于其它EGFR敏感突变，但对三代EGFR抑制剂奥希替尼敏感；再比如KRAS基因突变，它是影响TKI类靶向药物疗效的不利因素，只要有KRAS基因突变，常见的针对EGFR、ALK、ROS1等基因的靶向药物都无法使用，此外，具有KRAS基因突变的肺癌患者往往化疗药物效果不佳，使用培美曲塞、紫杉醇等一线药物联合铂类化疗效果要比没有KRAS突变的患者要差，但近几年来，多个靶 向或者免疫治疗药物，在KRAS突变患者身上展现了优秀的临床数据，尤其是KRAS G12C。AMG 510是近些年来第一个达到临床阶段的KRAS G12C抑制剂，AMG 510 I期研究的结果显示，AMG 510治疗KRAS突变的NSCLC(非小细胞肺癌)患者的总ORR(客观缓解率)为48％，DCR(疾病控制率)为96％，在13名接受剂量为960mg AMG 510的NSCLC患者中，ORR为54％，DCR为100％，即将正式开展临床试验。故在试剂盒设计时，这类位点就需要独立出来，作为独立一管进行检测，并且能够区分。
Figure PCTCN2021070914-appb-000001
Figure PCTCN2021070914-appb-000002
本发明的再一目的在于提供一种用于一次性检测多重肺癌基因突变的组合物，包括EGFR、KRAS、BRAF、HER2、ALK、ROS1、RET、NTRK1、NTRK2、NTRK3、MET等突变基因的引物、探针，上述引物和探针用两支PCR 8联管分组分装如表1：
表1肺癌突变特异性引物、新型探针及分配方式
Figure PCTCN2021070914-appb-000003
Figure PCTCN2021070914-appb-000004
Figure PCTCN2021070914-appb-000005
Figure PCTCN2021070914-appb-000006
Figure PCTCN2021070914-appb-000007
Figure PCTCN2021070914-appb-000008
Figure PCTCN2021070914-appb-000009
Figure PCTCN2021070914-appb-000010
进一步，本发明还提供了上述引物和探针的反应体系，包括：
(1)逆转录反应体系：纯化水5～25μL、5×逆转录缓冲液5～15μL、各引物20～150μmol、逆转录酶100～200U，总体积为20～30μL；
(2)PCR反应体系：纯化水15～45μL、10×PCR缓冲液5～15μL、MgCl 2 1～10mmol、各对引物1～20pmol、各对探针1～20pmol、dNTPs10～100pmol、Taq酶1～10U，总体积为30～50μL。
本发明的另一目的在于提供一种试剂盒，该试剂盒包括SEQ ID NO：1～281序列。具体地，该试剂盒包括两种PCR 8联管，两种PCR 8联管分别检测肺癌RNA基因融合和肺癌DNA基因突变。该试剂盒包含EGFR、KRAS、BRAF、HER2、ALK、ROS1、RET、NTRK1、NTRK2、NTRK3、MET等基因的特异性引物和特异探针，还包括PCR缓冲液、UNG酶等。
进一步，该PCR 8联管中检测肺癌RNA基因融合的检测试剂包括：ALK、NTRK1、NTRK2和NTRK3融合基因检测试剂各1管，ROS1融合基因检测试剂2管，MET 14号外显子跳跃基因突变检测试剂1管，RET融合基因检测试剂1管。该八联管中1～4号管分别由ALK、NTRK1、NTRK2和NTRK3融合基因检测试剂组成，5和6号管由ROS1融合基因检测试剂组成，7号管由MET 14号外显子跳跃突变检测试剂组成，8号管由RET融合基因检测试剂和内控检测试剂组成。1～8号管融合基因检测 试剂由FAM信号指示，1～8号管的内控检测试剂用于监控样本RNA质量及加入情况，由VIC信号指示。
该PCR 8联管中检测肺癌DNA基因突变的检测试剂包括：EGFR突变基因检测试剂3管、EGFR/BRAF突变基因检测试剂1管、EGFR/KRAS突变基因检测试剂1管、HER2/KRAS突变基因检测试剂2管，外控检测试剂1管。该八联管中1～5号管的FAM信号和1～3号管的ROX信号分别指示EGFR基因的不同突变位点，4号管的ROX信号指示BRAF基因的V600E突变，6～7号管的FAM信号指示HER2基因的不同突变位点，5～7号管的ROX信号分别指示KRAS基因的不同突变位点，1～7号管的内控检测试剂用于检测样本加入情况，由VIC信号指示，8号管由扩增人类基因组保守区段的检测试剂组成，用于监控样本DNA质量，由FAM和ROX信号指示。
检测一个样本需要2个PCR 8联管。试剂盒组成详见表2、表3和表4。
表2试剂盒组成
Figure PCTCN2021070914-appb-000011
表3 LET PCR 8联管-基因融合组成成分
管号 检测试剂 检测类型 体积 荧光信号
1 LET反应液A1 ALK融合 35μL FAM/VIC
2 LET反应液A2 NTRK1融合 35μL FAM/VIC
3 LET反应液A3 NTRK2融合 35μL FAM/VIC
4 LET反应液A4 NTRK3融合 35μL FAM/VIC
5 LET反应液A5 ROS1融合 35μL FAM/VIC
6 LET反应液A6 ROS1融合 35μL FAM/VIC
7 LET反应液A7 MET 14号外显子跳 35μL FAM/VIC
8 LET反应液A8 RET融合 35μL FAM/VIC
表4 LET PCR 8联管-基因突变组成成分
管号 检测试剂 检测类型 体积 荧光信号
1 LET反应液B1 EGFR突变 35μL FAM/VIC/ROX
2 LET反应液B2 EGFR突变 35μL FAM/VIC/ROX
3 LET反应液B3 EGFR突变 35μL FAM/VIC/ROX
4 LET反应液B4 EGFR/BRAF突变 35μL FAM/VIC/ROX
5 LET反应液B5 EGFR/KRAS突变 35μL FAM/VIC/ROX
6 LET反应液B6 HER2/KRAS突变 35μL FAM/VIC/ROX
7 LET反应液B7 HER2/KRAS突变 35μL FAM/VIC/ROX
8 LET反应液B8 外控 35μL FAM/ROX
表5 LET PCR 8联管-基因融合检测的位点
Figure PCTCN2021070914-appb-000012
Figure PCTCN2021070914-appb-000013
Figure PCTCN2021070914-appb-000014
Figure PCTCN2021070914-appb-000015
表6 LET PCR 8联管-基因突变检测的位点
Figure PCTCN2021070914-appb-000016
Figure PCTCN2021070914-appb-000017
Figure PCTCN2021070914-appb-000018
Figure PCTCN2021070914-appb-000019
本发明的再一目的在于提供该试剂盒的使用方法，其步骤包括：
(1)提取检测样本中的DNA和RNA，该检测样本包括新鲜病理组织、石蜡包埋组织或石蜡切片组织；
(2)将上述RNA与逆转录酶混合，分别加入逆转录反应液，得到cDNA；
(3)将上述DNA和该cDNA分别与混合酶混合，并加入到PCR 8联管；
(4)经荧光PCR扩增仪扩增后，根据显示的Ct值判断检测结果。
本发明的有益效果是：
1.本发明采用特异性引物和探针技术，三色荧光通道检测模式，建立了实时PCR 体系，可以同时检测ALK、ROS1、RET、NTRK1、NTRK2、NTRK3、MET七种融合基因和EGFR、KRAS、BRAF、HER2四种突变基因，共有119种基因融合和112种基因突变，具体包括，EGFR基因83种突变、KRAS基因7种突变、BRAF基因1种突变、HER2基因21种突变以及ALK基因46种融合、ROS1基因23种融合、RET基因18种融合、NTRK1基因18种融合、NTRK2基因5种融合、NTRK3基因8种融合和MET基因1种跳跃突变(详见表5、表6)。
2.本发明根据所检测的多个突变/融合位点的检测难易程度、引物之间的相互影响、竞争等多方面综合因素，将281条序列(包括引物和探针)分组，分装于两个PCR 8联管中，其优点在于：(1)采用两重或三重PCR，将多种突变类型并管，并用不同荧光标记的探针加以区分，可以实现在一管内检测多种突变类型，便于客户操作，同时节约了检测成本及检测样本；(2)在突变类型并管时，除了考虑技术上并管的难度，还兼顾不同药物的需求。对于同一种药物，尽量将药物相应靶点放置于同一个反应管中，或放置于不同反应管的同一荧光通道，便于结果分析；(3)试剂盒可一次性检测231种突变/融合位点，且各反应管的操作方法/扩增程序基本一致，操作简便，节约时间，便于临床应用；(4)每个反应体系中还含有内控检测体系，融合反应管的内控用于质控RNA样本的质量及操作、仪器是否异常；突变反应管的内控用于质控DNA样本的质量及操作、仪器是否异常，外控用于判断样本DNA质量，并参与结果判读。(5)灵敏度高，对1％的基因突变DNA和125拷贝的融合基因质粒DNA均可以检出；(6)检测时间短，检测过程只需要3个小时即可完成；(7)预分装设计，并进行密封处理，提高了运输过程的安全性，操作更方便。
3.本发明提供的试剂盒涵盖的检测位点均占到每种基因所有突变类型的95％以上，在非小细胞肺癌患者中总突变率高达70％，是目前检测突变基因最多、突变类型最完整的荧光PCR产品，为临床医生对肺癌患者选择肿瘤靶向药物治疗提供全面的参考。
4.与NGS方法相比，本发明提供的试剂盒操作简便快捷、灵敏度高、特异性好、结果判读客观等，成为临床普及性最高的一线检测方法。
附图说明
图1为实施例1中ALK融合基因—EML4 exon 13、ALK exon 20的灵敏度图。
图2为实施例1中ROS1融合基因—CD74 exon 6、ROS1 exon 32的灵敏度图。
图3为实施例1中RET融合基因—KIF5B exon 16、ROS1 exon 12的灵敏度图。
图4为实施例1中NTRK1融合基因—CD74 exon8、NTRK1 exon10的灵敏度图。
图5为实施例1中NTRK2融合基因—TRIM24 exon12、NTRK2 exon15的灵敏度图。
图6为实施例1中NTRK3融合基因—ETV6 exon5、NTRK3 exon15的灵敏度图。
图7为实施例1中MET跳跃突变—MET exon14 skipping的灵敏度图。
图8为实施例1中EGFR基因突变—19del的灵敏度图。
图9为实施例1中EGFR基因突变—L858R的灵敏度图。
图10为实施例1中EGFR基因突变—T790M的灵敏度图。
图11为实施例1中KRAS基因突变—G12D的灵敏度图。
图12为实施例1中KRAS基因突变—G12C的灵敏度图。
图13为实施例1中BRAF基因突变—V600E的灵敏度图。
图14为实施例1中HER2基因突变—V769_D770insASV的灵敏度图。
图15为实施例1中HER2基因突变—A775_G776insYVMA的灵敏度图。
图16为实施例2中临床阳性样本ALK融合基因检测结果的PCR图。
图17为实施例2中临床阳性样本ROS1融合基因检测结果的PCR图。
图18为实施例2中临床阳性样本RET融合基因检测结果的PCR图。
图19为实施例2中临床阳性样本NTRK3融合基因检测结果的PCR图。
图20为实施例2中临床阳性样本MET跳跃突变检测结果的PCR图。
图21为实施例2中临床阳性样本EGFR19del基因突变检测结果的PCR图。
图22为实施例2中临床阳性样本EGFR L858R基因突变检测结果的PCR图。
图23为实施例2中临床阳性样本EGFR T790M基因突变检测结果的PCR图。
图24为实施例2中临床阳性样本KRAS基因突变检测结果的PCR图。
图25为实施例2中临床阳性样本KRAS G12C基因突变检测结果的PCR图。
图26为实施例2中临床阳性样本BRAF基因突变检测结果的PCR图。
图27为实施例2中临床阳性样本HER2基因突变检测结果的PCR图。
以上附图中各图的横坐标均为循环数(cycles)，纵坐标均为荧光值Fluorescence(dR)。
具体实施方式
以下结合具体实施例对本发明进一步阐述，这些实施例仅用于解释本发明而不能 理解为对本发明的限制。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1
根据Cosmic数据公布的人类EGFR、BRAF、HER2、KRAS、ALK、ROS1、RET、NTRK1、NTRK2、NTRK3和MET野生型基因序列，以EGFR、BRAF、HER2、KRAS、ALK、ROS1、RET、NTRK1、NTRK2、NTRK3和MET的驱动性突变位点及融合位点为基础，来设计特异性引物和探针(详见表1、表7及表8)。
表7 LET A反应液检测位点分布
Figure PCTCN2021070914-appb-000020
Figure PCTCN2021070914-appb-000021
表8 LET反应液B检测位点分布
Figure PCTCN2021070914-appb-000022
Figure PCTCN2021070914-appb-000023
本实施例分别以ALK融合基因EML4 exon 13、ALK exon 20，ROS1融合基因CD74 exon 6、ROS1 exon 32，RET融合基因KIF5B exon 16、ROS1 exon 12，NTRK1融合基因CD74 exon8、NTRK1 exon10，NTRK2融合基因TRIM24 exon12、NTRK2  exon15，NTRK3融合基因ETV6 exon5、NTRK3 exon15，MET跳跃突变基因MET exon14 skipping，EGFR基因突变基因19del、L858R、T790M，KRAS突变基因G12D、G12C，BRAF基因突变基因V600E，HER2基因突变基因V769_D770insASV、A775_G776insYVMA为例来阐述本发明的荧光PCR检测上述肺癌基因。
实验分别用含上述融合基因的T7RNA及各突变的质粒模板，其荧光PCR检测包括以下步骤：
(一)质粒、T7RNA处理与提取：
质粒采用天根生化的质粒小提试剂盒(货号：DP103)进行DNA提取，将质粒采用TranscriptAid T7 High Yield Transcription Kit(货号：K0441)转录获得T7 RNA，具体操作步骤按试剂盒说明书操作。DNA和RNA提取结束后，立即使用紫外分光光度计检测DNA和RNA的浓度及纯度。DNA和RNA的OD 260/OD 280应介于1.7～2.1；RNA浓度应介于10～100ng/μL。
(二)建立PCR扩增反应体系：
将上述所得模板，用作实时荧光PCR扩增的模板，按照如下扩增体系进行PCR扩增：
逆转录反应体系：纯化水5～25μL、5×逆转录缓冲液5～15μL、各引物20～150μmol、逆转录酶100～200U，总体积为20～30μL；
PCR反应体系：纯化水15～45μL、10×PCR缓冲液5～15μL、MgCl 2 1～10mmol、各对引物1～20pmol、各对探针1～20pmol、dNTPs10～100pmol、Taq酶1～10U，总体积为30～50μL。
逆转录反应条件：42℃60min，95℃5min，1个循环；
得到S-cDNA1和S-cDNA2，与酶混合后，分别加入到检测管LET PCR 8联管-基因融合-1～4和检测管LET PCR 8联管-基因融合-5～8；DNA样本则加入检测管LET PCR 8联管-基因突变-1～8中，进行PCR扩增。
实时PCR反应条件为：
第一阶段：42℃5min，95℃5min，1个循环；
第二阶段：95℃25s，64℃20s，72℃20s，10个循环；
第三阶段：93℃25s，60℃35s，72℃20s，36个循环；
信号收集：第三阶段60℃时收集FAM/VIC/ROX信号，执行实时PCR，保存文件。
(三)检测结果的判断：根据荧光PCR扩增仪显示的Ct值判断检测结果(详见表9、表10)。
1.确认未选择校正荧光参照，按管号顺序依次选择单一检测反应管进行分析，同时选择阳性对照反应管、样本反应管和阴性对照管，然后用户可根据实际情况确定扩增曲线升起的拐点处，得到各反应管的Ct值。
2.阴性对照FAM、VIC和ROX信号扩增曲线：
阴性对照LET PCR 8联管-基因融合-1～8FAM信号和LET PCR 8联管-基因突变-1～7FAM和ROX信号应无扩增曲线升起，若任意一管上述信号有扩增曲线升起，则此次实验结果无效，建议重新检测。
3.阳性对照FAM、VIC和ROX信号扩增曲线：
①阳性对照LET PCR 8联管-基因融合-1～8和LET PCR 8联管-基因突变-1～8的FAM信号应全部都有扩增曲线升起，且Ct值一般小于25，但可能会由于不同阈值设置而发生波动。
②阳性对照LET PCR 8联管-基因突变-1～8的ROX信号应全部都有扩增曲线升起，且Ct值小于25。
③阳性对照的LET PCR 8联管-基因融合-1～8和LET PCR 8联管-基因突变-1～7的VIC信号应全部都有扩增曲线升起，且Ct值小于25。
4.样本RNA基因融合阴阳性判定：
①样本LET PCR 8联管-基因融合-1～8的VIC信号扩增曲线：
a.若8个管Ct值均＜36且任意一管Ct值＜26，则继续分析；
b.若8个管Ct值均≥26或任意一管Ct值≥36，说明RNA可能存在片段化或RNA降解或样本存在抑制剂或漏加，建议重新检测或重新提取RNA后再进行检测。
②样本LET PCR 8联管-基因融合-1～8的FAM信号扩增曲线：
a.若任意一管FAM信号Ct值落在阳性区域(详见表9)，则该样本判定为该反应管阳性；
b.若所有管FAM信号Ct值均落在阴性区域(详见表9)，则该样本判定为阴性或低于本试剂盒的检测下限。
表9 LET PCR 8联管-基因融合FAM信号结果判定
Figure PCTCN2021070914-appb-000024
Figure PCTCN2021070914-appb-000025
注意：5号管阳性样本可能会在6号管出现交叉反应扩增，因此，当5和6号管都存在阳性扩增曲线时，判定5号管为真阳性扩增信号，6号管为交叉反应扩增信号。
5.样本DNA基因突变阴阳性判定：LET PCR 8联管-基因突变中，首先确定样本1～7号管FAM和ROX信号各自的Ct值，然后确定该样本8号管FAM和ROX信号的Ct值。由于样本中突变百分含量各不相同，所得到的突变Ct值也各不相同。根据不同的突变Ct值，把样本检测结果分为阴性、阳性A区及阳性B区。具体判定详见表10。
①样本LET PCR 8联管-基因突变1～7号管VIC信号扩增曲线：
a.若1～7号管Ct值均＜36，则继续分析；
b.若1～7号管任意一管Ct值≥36，说明DNA可能存在片段化或DNA降解或样本存在抑制剂或漏加，建议重新检测或重新提取DNA后再进行检测。
②样本LET PCR 8联管-基因突变1～8号管FAM和ROX信号扩增曲线：
a.若19≤8号管FAM和ROX信号的Ct值≤25，则继续分析；
b.若8号管FAM和ROX信号的Ct值＜19，说明加入的DNA过量，应减少DNA加入量再进行实验。但突变信号无升起或者落在阴性区，该实验结果仍然可信；
表10 LET PCR 8联管-基因突变FAM/ROX信号结果判定
Figure PCTCN2021070914-appb-000026
c.若8号管FAM和ROX信号的Ct值＞25，说明加入的DNA可能存在片段化或降解或存在抑制剂或漏加，若突变信号有升起且落在阳性A区，实验结果仍然可信，否则建议增加DNA上样量或重新提取DNA后再进行检测；
d.当样本1～7号管FAM和ROX信号各管的Ct值都大于或等于阴性临界值时(即落于阴性区)，则该样本为阴性或低于本试剂盒的检测下限；
e.当样本1～7号管FAM和ROX信号的某个反应管Ct值小于阴性临界值时，进行下列判断：
i.当某个反应管的Ct值小于阳性临界值时(即落于阳性A区)，则该样本为该反应管对应的突变阳性；
ii.当某个反应管的Ct值大于阳性临界值时，且小于阴性临界Ct值时(即落于阳性B区)，则计算该反应管的ΔCt值。若反应管的ΔCt值小于相对应的ΔCt Cut-off值，则该样品也为该反应管对应的突变阳性，即阳性B区；反之则为阴性或低于本试剂盒的检测下限。
③ΔCt值的计算：ΔCt值＝突变FAM(ROX)Ct值-外控FAM(ROX)Ct值。突变FAM(ROX)Ct值是指样本突变信号1～7号管(FAM或ROX信号)对应的Ct值；外控Ct值是指样本对应的外控信号8号管(FAM或ROX信号)的Ct值。
6.某些EGFR、HER2、KRAS阳性样品可能会导致个别突变反应管之间出现交叉信号。当样品在2个或2个以上反应管出现阳性结果(按照表10判读)时，先确定突变反应管中Ct值最小的为真阳性，再计算各反应管的ΔCt值(突变Ct值-外控Ct值)，并按照表11～13所列交叉信号阈值来判定其它反应管是否为交叉信号。
①若ΔCt值小于交叉信号阈值，则认为是真阳性信号，可判为该突变反应管阳性；
②若ΔCt值大于或等于交叉阈值，则认为是交叉信号，可判为该突变反应管阴性。
表11 EGFR交叉反应阈值表
Figure PCTCN2021070914-appb-000027
表12 HER2交叉反应阈值表
Figure PCTCN2021070914-appb-000028
表13 KRAS交叉反应阈值表
Figure PCTCN2021070914-appb-000029
Figure PCTCN2021070914-appb-000030
7.一个样本可能同时含有2个或多个融合或突变类型。
(四)灵敏度分析：分别取DNA和RNA的质粒进行梯度稀释，每种基因最终均有至少2种浓度进行检测限考察。结果表明本发明的荧光PCR方法的灵敏度高，1％的基因突变DNA和25拷贝/μL的融合基因质粒均可检出，部分结果如图1～图15。
检测结果表明，本发明的检测体系可以一次性准确检测肺癌11种基因，检测的灵敏度可达到1％的基因突变DNA和25拷贝/μL的融合基因RNA。
实施例2
运用本发明检测临床样本，2019年11月对172例临床NSCLC FFPE样本进行肺癌11种基因突变/融合的检测(包括部分已知结果的临床样本)，并与NGS方法进行对比。
一、检测样本DNA和RNA的提取：DNA和RNA的提取使用厦门艾德生物医药科技股份有限公司的核酸提取试剂(型号：FFPE DNA/RNA，医疗器械备案号：闽厦械备20150082号，货号：8.0223601X036G)。DNA和RNA提取结束后，使用微量紫外分光光度计检测DNA和RNA的浓度与纯度。RNA浓度应大于10ng/μL，DNA浓度应大于2ng/μL，DNA和RNA的OD 260/OD 280应介于1.7～2.1之间。将上述提取的DNA和RNA用作肺癌11种基因检测的扩增模板。
二、将上述所提的DNA和RNA，分别按照如下扩增体系进行PCR扩增：
逆转录反应体系：纯化水5～25μL、5×逆转录缓冲液5～15μL、各引物20～150μmol、逆转录酶100～200U，总体积为20～30μL；
PCR反应体系：纯化水15～45μL、10×PCR缓冲液5～15μL、MgCl2 1～10mmol、各对引物1～20pmol、各对探针1～20pmol、dNTPs10～100pmol、Taq酶1～10U，总体积为30～50μL。逆转录反应条件：42℃60min，95℃5min，1个循环；
得到S-cDNA1和S-cDNA2，与酶混合后，分别加入到检测管LET PCR 8联管-基因融合-1～4和5～8；DNA样本则加入检测管LET PCR 8联管-基因突变-1～8中，进行PCR扩增。
实时PCR反应条件为：
第一阶段：42℃5min，95℃5min，1个循环；
第二阶段：95℃25s，64℃20s，72℃20s，10个循环；
第三阶段：93℃25s，60℃35s，72℃20s，36个循环；
信号收集：第三阶段60℃时收集FAM/VIC/ROX信号，执行实时PCR，保存文件。
三、检测结果的判断：根据荧光PCR扩增仪显示的Ct值判断检测结果(判断方法参照实施例1)。
四、NGS检测。提取合格的DNA样本，片段化后进行末端修复及接头连接，采用艾德人泛肿瘤116基因panel捕获探针，进行目标区域的捕获，质控合格的捕获后文库采用illumina Novaseq 6000测序平台进行测序，并用艾德人泛癌种116基因突变联合检测数据分析系统(ADXPAN116-tMut_v0.2.0)对测序数据进行分析，得到116种基因(包括EGFR、KRAS、BRAF、HER2、ALK、ROS1、RET、NTRK1、NTRK2、NTRK3、MET等)的基因变异结果。
172例临床样本，本发明试剂盒共检测出125例阳性样本，样本突变情况分布详见表11、表12和图16～27。
表11临床样本的突变情况分布
突变名称 本发明(例数) NGS(例数)
EGFR突变阳性 73 72
KRAS突变阳性 13 13
BRAF突变阳性 3 3
HER2突变阳性 1 1
ALK融合阳性 11 11
ROS1融合阳性 8 8
NTRK3融合阳性 4 4
RET融合阳性 7 7
MET Skipping阳性 7 7
表12本发明与NGS检测结果四格表分析
Figure PCTCN2021070914-appb-000031
1例不一致样本采用已上市的人类EGFR基因突变检测试剂盒(荧光PCR法)(国械注准20143402001)进行验证，结果为EGFR 19del阳性，与本发明检测结果一致。
两种方法检测的数据分析如下：
阳性符合率＝124/124＝100.00％(95％CI：97.64％，100.76％)；
阴性符合率＝47/48＝97.92％(95％CI：93.88％，101.96％)；
总符合率＝171/172＝99.42％(95％CI：98.28％，100.55％)。
Kappa值＝0.985，标准误S K＝0.0145，Z值＝67.965，根据Z值，以ν＝∞查t界值表，t临界值是1.96(双侧0.05)或者1.64(单侧0.05)，Z＝67.965＞1.96，P＜0.05，可认为Kappa值具统计学意义，两种试剂盒的检测结果存在一致性。
该荧光PCR方法和NGS结果的总符合率高达99.42％，荧光PCR灵敏度和选择性检测能力与NGS相当。与NGS相比，PCR方法检测11种基因方便快捷，耗时短，节约样本，更有利于临床推广使用。
以上所述，仅为本发明的较佳实施例而已，故不能依此限定本发明实施的范围，即依本发明专利范围及说明书内容所作的等效变化与修饰，皆应仍属本发明涵盖的范围内。
工业实用性
本发明公开了一种性检测肺癌多重基因突变的试剂盒，其中包括用于检测肺癌EGFR、KRAS、BRAF、HER2、ALK、ROS1、RET、NTRK1、NTRK2、NTRK3、MET基因突变的引物、探针及其分配方式。本发明采用PCR 8联管设计，每两个PCR 8联管检测一个样品的11种基因突变/融合状态，其中一个PCR 8联管内装有相应的融合检测试剂和内控试剂；另一个PCR 8联管管内装有相应的突变检测试剂。本发明应用荧光PCR方法实现一次性检测肺癌的119种基因融合和112种基因突变类型，大大缩短了检测时间，操作简便，结果准确,具有工业实用性。

Claims (19)

  1. 一种检测肺癌基因突变的试剂盒，其特征在于，所述的试剂盒包括：
    用于检测肺癌基因融合的A1组位点的试剂，所述A1组位点包括EML4-ALK-1至EML4-ALK-3、EML4-ALK-6至EML4-ALK-14、EML4-ALK-17至-EML4-ALK-21；KIF5B-ALK-1；KIF5B-ALK-2；KLC1-ALK；TFG-ALK；STRN-ALK-1；HIP1-ALK-1；HIP1-ALK-2；HIP1-ALK-3；PRKAR1A-ALK-1；PRKAR1A-ALK-2；NBAS-ALK-1；TFG-ALK-2；PPM1B-ALK-1；EIF2AK3-ALK-1；BCL11A–ALK-1；BIRC6-ALK-1；CEBPZ-ALK-1；CLIP1-ALK-1；COL25A1-ALK-1；GCC2-ALK-1；GCC2-ALK-2；LMO7-ALK-1；PICALM-ALK-1；PHACTR1-ALK-1；TPR-ALK-1；MPRIP-ALK-1；TNIP2-ALK-1；DCTN1-ALK-1；SQSTM1-ALK-1；
    用于检测肺癌基因融合A5组位点的试剂，所述第A5组位点包括ROS1-M1至ROS1-M10；
    用于检测肺癌突变基因B1组位点的试剂，所述B1组位点包括E-19-M1至E-19-M21；E-19-M24至E-19-M27；E-20-M2；
    用于检测肺癌突变基因B2组位点的试剂，所述B2组位点包括E-21-M1；E-18-M1；E-18-M2；E-18-M3；
    用于检测肺癌突变基因B3组位点的试剂：所述B3组位点包括E-20-M1；E-21-M2。
  2. 如权利要求1所述的一种检测肺癌基因突变的试剂盒，其特征在于，所述试剂盒的反应体系，包括：
    (1)逆转录反应体系：纯化水5～25μL、5×逆转录缓冲液5～15μL、各引物20～150μmol、逆转录酶100～200U，总体积为20～30μL；
    (2)PCR反应体系：纯化水15～45μL、10×PCR缓冲液5～15μL、MgCl 21～10mmol、各对引物1～20pmol、各对探针1～20pmol、dNTPs10～100pmol、Taq酶1～10U，总体积为30～50μL。
  3. 根据权利要求1所述的一种检测肺癌基因突变的试剂盒，其特征在于，所述的试剂组合包括如下引物和探针：用于检测肺癌基因融合的A1组位点的试剂组合：SEQ ID NO：1-SEQ ID NO：44。
  4. 根据权利要求1所述的一种检测肺癌基因突变的试剂盒，其特征在于，所述的试剂组合包括如下引物和探针：用于检测肺癌基因融合的A5组位点的试剂组合，：SEQ ID NO：92-SEQ ID NO：104。
  5. 根据权利要求1所述的一种检测肺癌基因突变的试剂盒，其特征在于，所述的试剂组合包括如下引物和探针：用于检测肺癌基因融合的B1组位点的试剂组合：SEQ ID NO：155-SEQ ID NO：174。
  6. 根据权利要求1所述的一种检测肺癌基因突变的试剂盒，其特征在于，所述的试剂组合包括如下引物和探针：用于检测肺癌基因融合的B2组位点的试剂组合，：SEQ ID NO：175-SEQ ID NO：187。
  7. 根据权利要求1所述的一种检测肺癌基因突变的试剂盒，其特征在于，所述的试剂组合包括如下引物和探针：用于检测肺癌基因融合的B3组位点的试剂组合，：SEQ ID NO：188-SEQ ID NO：198。
  8. 一种检测肺癌基因突变的试剂盒，包括15个组合，其中：
    用于检测如下A1组位点的试剂：包括EML4-ALK-1至EML4-ALK-3、EML4-ALK-6至EML4-ALK-14、EML4-ALK-17至-EML4-ALK-21；KIF5B-ALK-1；KIF5B-ALK-2；KLC1-ALK；TFG-ALK；STRN-ALK-1；HIP1-ALK-1；HIP1-ALK-2；HIP1-ALK-3；PRKAR1A-ALK-1；PRKAR1A-ALK-2；NBAS-ALK-1；TFG-ALK-2；PPM1B-ALK-1；EIF2AK3-ALK-1；BCL11A–ALK-1；BIRC6-ALK-1；CEBPZ-ALK-1；CLIP1-ALK-1；COL25A1-ALK-1；GCC2-ALK-1；GCC2-ALK-2；LMO7-ALK-1；PICALM-ALK-1； PHACTR1-ALK-1；TPR-ALK-1；MPRIP-ALK-1；TNIP2-ALK-1；DCTN1-ALK-1；SQSTM1-ALK-1；
    用于检测如下A2组位点的试剂：包括NTRK1-E9-M1；NTRK1-E10-M1；NTRK1-E10-M3；NTRK1-E10-M5至NTRK1-E10-M9；NTRK1-E10-M12；NTRK1-E10-M14；NTRK1-E10-M15；NTRK1-E10-M17；NTRK1-E12-M1；NTRK1-E12-M3；NTRK1-E12-M4；NTRK1-E12-M11；NTRK1-E12-M12；NTRK1-E12-M14；
    用于检测如下A3组位点的试剂：包括NTRK2-E15-M1；NTRK2-E16-M1；NTRK2-E16-M3；NTRK2-E16-M7；NTRK2-E17-M2；
    用于检测如下A4组位点的试剂：包括NTRK3-EX14-M1；NTRK3-EX14-M2；NTRK3-EX14-M3；NTRK3-EX14-M4；NTRK3-EX14-M7；NTRK3-EX15-M1；NTRK3-EX15-M2；NTRK3-EX15-M3；
    用于检测如下A5组位点的试剂：包括ROS1-M1至ROS1-M10；
    用于检测如下A6组位点的试剂：包括ROS1-M11；ROS1-M12；ROS1-M13；ROS1-EX35-M1至ROS1-EX35-M4；ROS1-EX35-M6；ROS1-EX35-M8至ROS1-EX35-M11；ROS1-EX35-M13；
    用于检测如下A7组位点的试剂：包括MET-M1；
    用于检测如下A8组位点的试剂：包括RET–M2；RET–M5；RET-M15；RET–M16；RET–M17；RET–M19；LRET-M22；LRET-M32；LRET-M40；LRET-M41；LRET-M42；LRET-M44；LRET-M45；LRET-M49；LRET-M55；LRET-M57；LRET-M58；LRET-M59；
    用于检测如下B1组位点的试剂：包括E-19-M1至E-19-M21；E-19-M24至E-19-M27；E-20-M2；
    用于检测如下B2组位点的试剂：包括E-21-M1；E-18-M1；E-18-M2；E-18-M3；
    用于检测如下B3组位点的试剂：包括E-20-M1；E-21-M2；
    用于检测如下B4组位点的试剂：包括E-20-M4；E-20-M5；E-20-M8；E-20-M9；E-20-M13；E-20-M14；E-20-M20；E-20-M21；E-20-M23；E-20-M24；E-20-M28至E-20-M31；E-20-M36至E-20-M41；E-20-M44；E-20-M48；E-20-M50至E-20-M53；BRAF-M1；
    用于检测如下B5组位点的试剂：包括E-20-M3；E-20-M10；E-20-M12；E-20-M15至E-20-M19；E-20-M22；E-20-M25至E-20-M27；E-20-M32至E-20-M35；E-20-M42；E-20-M43；E-20-M45至E-20-M47；E-20-M49；E-20-M55至E-20-M57；KRAS-M6；
    用于检测如下B6组位点的试剂：包括HER2-M1至HER2-M3；HER2-M8；HER2-M9；HER2-M15至HER2-M26；KRAS-M2；KRAS-M3；KRAS-M5；KRAS-M14；
    用于检测如下B7组位点的试剂：包括HER2-M4；HER2-M6；HER2-M7；HER2-M10；KRAS-M1；KRAS-M4。
  9. 如权利要求8所述的一种检测肺癌基因突变的试剂盒，其特征在于，所述试剂盒的反应体系，包括：
    (1)逆转录反应体系：纯化水5～25μL、5×逆转录缓冲液5～15μL、各引物20～150μmol、逆转录酶100～200U，总体积为20～30μL；
    (2)PCR反应体系：纯化水15～45μL、10×PCR缓冲液5～15μL、MgCl 21～10mmol、各对引物1～20pmol、各对探针1～20pmol、dNTPs10～100pmol、Taq酶1～10U，总体积为30～50μL。
  10. 根据权利要求8所述的一种检测肺癌基因突变的试剂盒，其特征在于，所述的试剂组合包括如下引物和探针：
    用于检测肺癌基因融合的A1组位点的试剂：SEQ ID NO：1-SEQ ID NO：44；
    用于检测肺癌基因融合的A2组位点的试剂：SEQ ID NO：45-SEQ ID NO：66；
    用于检测肺癌基因融合的A3组位点的试剂：SEQ ID NO：67-SEQ ID NO：78；
    用于检测肺癌基因融合的A4组位点的试剂：SEQ ID NO：79-SEQ ID NO：91；
    用于检测肺癌基因融合的A5组位点的试剂，：SEQ ID NO：92-SEQ ID NO：104；
    用于检测肺癌基因融合的A6组位点的试剂：SEQ ID NO：105-SEQ ID NO：122；
    用于检测肺癌基因融合的A7组位点的试剂：SEQ ID NO：123-SEQ ID NO：131；
    用于检测肺癌基因融合的A8组位点的试剂：SEQ ID NO：132-SEQ ID NO：154；
    用于检测肺癌基因融合的B1组位点的试剂：SEQ ID NO：155-SEQ ID NO：174；
    用于检测肺癌基因融合的B2组位点的试剂：SEQ ID NO：175-SEQ ID NO：187；
    用于检测肺癌基因融合的B3组位点的试剂：SEQ ID NO：188-SEQ ID NO：198。
    用于检测肺癌基因融合的B4组位点的试剂：SEQ ID NO：199-SEQ ID NO：220；
    用于检测肺癌基因融合的B5组位点的试剂：SEQ ID NO：221-SEQ ID NO：244；
    用于检测肺癌基因融合的B6组位点的试剂：SEQ ID NO：245-SEQ ID NO：262；
    用于检测肺癌基因融合的B7组位点的试剂：SEQ ID NO：263-SEQ ID NO：273。
  11. 根据权利要求8所述的一种检测肺癌基因突变的试剂盒，其特征在于，
    还包括用于质控样本质量的外控试剂组合。
  12. 根据权利要求11所述的一种检测肺癌基因突变的试剂盒，其特征在于，所述的试剂组合包括如下引物和探针：SEQ ID NO：274-SEQ ID NO：281。
  13. 一种用于检测肺癌多重基因突变的组合物，其特征在于，该组合物包括下表所示的281条序列的引物和探针，且该281条序列用两支PCR 8联管分组分装如下：
    Figure PCTCN2021070914-appb-100001
    Figure PCTCN2021070914-appb-100002
    Figure PCTCN2021070914-appb-100003
    Figure PCTCN2021070914-appb-100004
    Figure PCTCN2021070914-appb-100005
    Figure PCTCN2021070914-appb-100006
    Figure PCTCN2021070914-appb-100007
    Figure PCTCN2021070914-appb-100008
    Figure PCTCN2021070914-appb-100009
  14. 根据权利要求13所述的组合物，其特征在于，所述的引物和探针的反应体系，包括：
    (1)逆转录反应体系：纯化水5～25μL、5×逆转录缓冲液5～15μL、各引物20～150μmol、逆转录酶100～200U，总体积为20～30μL；
    (2)PCR反应体系：纯化水15～45μL、10×PCR缓冲液5～15μL、MgCl 21～10mmol、各对引物1～20pmol、各对探针1～20pmol、dNTPs10～100pmol、Taq酶1～10U，总体积为30～50μL。
  15. 一种试剂盒，包括权利要求13中所述的281条序列的引物和探针。
  16. 根据权利要求15所述的一种试剂盒，其特征在于，所述试剂盒包括两种PCR 8联管，该两种PCR 8联管分别检测肺癌RNA基因融合和肺癌DNA基因突变。
  17. 根据权利要求15或16所述的一种试剂盒，其特征在于，所述PCR 8联管中检测肺癌RNA基因融合的检测试剂包括：ALK、NTRK1、NTRK2和NTRK3融合基因检测试剂各1管，ROS1融合基因检测试剂2管，MET 14号外显子跳跃基因突变检测试剂1管，RET融合基因检测试剂1管。
  18. [根据细则91更正 17.03.2021]　
    根据权利要求15或16所述的一种试剂盒，其特征在于，所述PCR 8联管中检测肺癌DNA基因突变的检测试剂包括：EGFR、EGFR、EGFR、EGFR/BRAF、EGFR/KRAS、HER2/KRAS、HER2/KRAS突变基因检测试剂各1管，外控检测试剂1管。
  19. [根据细则91更正 17.03.2021]
    权利要求15至18中任一权利要求所述的一种试剂盒的使用方法，其特征在于，包括：
    (1)提取检测样本中的DNA和RNA，所述检测样本包括新鲜病理组织、石蜡包埋组织或石蜡切片组织；
    (2)将所述RNA与逆转录酶混合，分别加入逆转录反应液，得到cDNA；
    (3)将所述DNA和所述cDNA分别与混合酶混合，并加入到PCR 8联管；
    (4)经荧光PCR扩增仪扩增后，根据显示的Ct值判断检测结果。
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