A primer composition and reagents for fusion gene detection

Abstract

The application discloses a primer composition and a reagent for fusion gene detection, and can detect human lung cancer fusion genes based on a multiplex PCR method. The primer composition can specifically amplify at least 50 fusion mutation forms of five fusion driver genes, such as ALK, NRG, NTRK1, RET and ROS1, in an RNA sample through sequences shown in SEQ ID NO 1-54, and can serve as a housekeeping gene for sample quality control. The reaction can be carried out in one tube or can be split into multiple tubes for carrying out. In the multiple tube reaction, a specific probe composition modified with a fluorescent group and having sequences shown in SEQ ID NO 55-70 can be added to form a qPCR reagent. When the qPCR reagent is used to detect a positive sample, the fusion gene in 0.04 ng of RNA can be reported. The process of the application is simple, efficient, easy to operate and low in cost, and has a good prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of gene detection, in particular to a primer composition and a reagent for fusion gene detection. BACKGROUND

[0002] When a tumor occurs, breakage and re-ligation at the genome level often occur. When two genes are broken into two halves respectively and the wrong mutual ligation occurs, a new gene, i.e. a fusion gene, can be formed. When the oncogenic fusion gene is transcribed into mRNA, its structure can be divided into two parts: the 3' end is usually from a gene with kinase activity, which originally stimulates cell growth and proliferation, such as ALK, RET, etc. These genes are called "driver genes" of fusion gene mutations; the sequence at the 5' end is more diverse, and the source gene is also called "partner gene" of fusion gene mutation. The common feature of these sequences is that they have high and difficult-to-regulate expression in tumor cells, resulting in excessive kinase activity stimulating growth, continuous activation of downstream regulatory pathways, and ultimately leading to carcinogenesis.

[0003] Studies have shown that about 10% of lung cancer is caused by fusion gene mutations, among which the most common is ALK fusion, followed by ROS1 fusion, and other fusions include RET fusion, NTRK fusion, NRG fusion, etc. There are already targeted drugs such as ceritinib, crizotinib, and entrectinib on the market that are specifically designed for fusion gene mutations. The NCCN guidelines and CSCO guidelines also clearly state that ALK fusion and ROS1 fusion detection is recommended for lung cancer patients, and the related detection market is very large.

[0004] Currently, the main methods for fusion gene detection are FISH (fluorescence in situ hybridization), IHC (immunohistochemistry), qPCR (quantitative PCR), NGS (next-generation sequencing), etc. FISH is the current gold standard for fusion detection, but its sensitivity is relatively low (10-15%), it is time-consuming and complex to operate, and it also relies on manual judgment. The advantage of IHC is that it is relatively low in price, but the judgment standard is more subjective, and it cannot directly detect fusion gene fragments. qPCR has high sensitivity (1-5%), is easy to operate, and is relatively low in price, but it can only detect one or a few known fusion mutations at a time. NGS has slightly lower sensitivity than qPCR, and can detect a large number of fusion mutations at a time if designed properly, but it still has the problems of long time consumption and high detection cost. In summary, the existing detection methods all have certain limitations and need to be further improved. SUMMARY

[0005] The technical problem solved by the present application is to provide a primer composition and a reagent for fusion gene detection, which can detect human lung cancer fusion genes based on a multiplex PCR method.

[0006] To solve the above technical problems, one technical solution of the present application is to provide a primer composition, wherein the primer in the primer composition comprises part or all of the sequences shown in SEQ ID NO. 1-52, and can specifically amplify the fusion gene fragment.

[0007] In a preferred embodiment of the present application, the primer comprises part or all of the sequences selected from 18-25 bases in each of the sequences shown in SEQ ID NO. 1-52, and can specifically amplify the fusion gene fragment.

[0008] In a preferred embodiment of the present application, the primer in the primer composition further comprises the sequences shown in SEQ ID NO. 53-54, and can specifically amplify the housekeeping gene fragment.

[0009] In a preferred embodiment of the present application, the primer comprises 18-25 bases in each of the sequences shown in SEQ ID NO. 53-54, and can specifically amplify the housekeeping gene fragment.

[0010] A reagent for fusion gene detection, comprising the primer composition described above.

[0011] In a preferred embodiment of the present application, the detectable fusion form is part or all of the following fusion forms in the table:

[0012]

[0013]

[0014] In a preferred embodiment of the present application, the reagent for fusion gene detection further comprises a probe composition for detecting the amplification product of the fusion gene, wherein the probe sequence in the probe composition comprises the sequences shown in SEQ ID NO. 55-70.

[0015] In a preferred embodiment of the present application, the probe sequence in the probe composition comprises part or all of the sequences selected from 20-32 bases in each of the sequences shown in SEQ ID NO. 55-70.

[0016] In a preferred embodiment of the present application, the 5' end of the probe in the probe composition can be connected to a fluorescent reporter group, and the 3' end of the probe can be selectively connected to a fluorescent quencher group.

[0017] In a preferred embodiment of the present application, the reagent for fusion gene detection is used in the detection of fusion genes from RNA samples from fresh or frozen tissues, whole blood, and FFPE.

[0018] In a preferred embodiment of the present application, the reagent for fusion gene detection is used in fusion gene detection based on qPCR or NGS method.

[0019] The present application has the following advantages:

[0020] Firstly, the primer composition of the present application can detect at least 50 fusion gene forms of 5 common fusion driver genes, such as ALK, NRG, NTRK1, RET and ROS1, in an RNA sample at one time, which meets the general needs of lung cancer tumor auxiliary diagnosis, drug use and prognosis.

[0021] Secondly, the primer composition of the present application can detect all fusion forms in the same tube reaction, or can be split to detect different fusion forms according to the needs, to adapt to the needs of different reporting methods. When using one tube reaction, the fusion can be reported by NGS in the subsequent process, which has the advantages of short overall time consumption and low cost compared with the general NGS fusion detection method; when using separate tube reactions and combining with the probe composition of the present application, the fusion can be reported by qPCR, which has the characteristics of more fusion forms that can be detected and high sensitivity, and can detect fusion genes as low as 0.04 ng of sample.

[0022] Thirdly, the method of the present application is simple and has low overall cost, which fills the gap of domestic multiple PCR technology for detecting fusion genes. The present application is helpful to realize the transformation of reagents from laboratory to clinical application and in vitro diagnostic reagents, and has good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0024] Figure 1 is the detection result graph of H2228 cell strain ALK fusion and housekeeping gene in the second embodiment of the present application;

[0025] Figure 2 is the detection result graph of HCC78 cell strain ROS1 fusion and housekeeping gene in the second embodiment of the present application. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] In the following examples, RNA was extracted from cell line samples using the Qiagen RNeasy Mini Kit (catalog number: 74104); RNA was extracted from FFPE samples using the Magen HiPure FFPE RNA Plus Kit (catalog number: R4144); and the extracted RNA was quantified using the Thermo Fisher Qubit dsRNA HS Assay Kit (catalog number: Q32852 / Q32855).

[0028] Because the fluorescence channels of the real-time PCR instrument are limited, the probe compositions are divided into two groups to distinguish between different fusion driver genes and housekeeping genes, as shown in the table below. Unless otherwise specified, each sample is also tested in two tubes.

[0029]

[0030]

[0031] Example 1:

[0032] I. Sample Preparation

[0033] Total RNA was extracted from each cell line shown in the table below and quantified using Qubit.

[0034] Cell lines Fusion forms H2228 EML4-ALK KM12 TPM3-NTRK1 LC-2 / ad CCDC6-RET HCC78 SLC34A2-ROS1 A549 None (negative)

[0035] II. Experimental Procedure:

[0036] (1) Prepare qPCR reaction solution as shown in the table below, and add 4 RNA samples to each tube.

[0037]

[0038] (2) Cover the tube, invert and mix well to avoid generating air bubbles, and then centrifuge quickly.

[0039] (3) Run the qPCR reaction according to the procedure in the table below.

[0040]

[0041]

[0042] III. Test Results

[0043] (1) The following table is the Ct value of tube 1 detection, and U.D in the table means Undetermined.

[0044] Fluorescence channel FAM HEX Texas Red Cy5 Corresponding gene ALK NTRK1 NRG Housekeeping gene H2228-1 27.79 U.D U.D 25.66 H2228-2 27.67 U.D U.D 25.61 H2228 average 27.73 U.D U.D 25.64 KM12-1 U.D 30.04 U.D 27.45 KM12-2 U.D 30.03 U.D 27.76 KM12 average U.D 30.04 U.D 27.61 LC-2 / ad-1 U.D U.D U.D 25.25 LC-2 / ad-2 U.D U.D U.D 25.44 LC-2 / ad average U.D U.D U.D 25.35 HCC78-1 U.D U.D U.D 27.14 HCC78-2 U.D U.D U.D 27.13 HCC78 average U.D U.D U.D 27.13 A549-1 U.D U.D U.D 27.85 A549-2 U.D U.D U.D 27.5 A549 average U.D U.D U.D 27.68

[0045] (2) The following table is the Ct value of tube 2 detection.

[0046]

[0047]

[0048] Four, result explanation

[0049] (1) The RNA of fusion negative cell strain can only detect housekeeping genes, without non-specific fusion results.

[0050] (2) The RNA of four fusion positive cell strains can detect corresponding fusion and housekeeping genes, and there is no non-specific fusion detection.

[0051] Example two

[0052] I. Sample preparation

[0053] The total RNA of cell strains H2228 and HCC78 was extracted respectively, and quantified by Qubit.

[0054] The total RNA of two cell strains was diluted by 4 times gradient respectively, forming a series of solutions containing 40, 10, 2.5, 0.63, 0.16, 0.04 ng RNA respectively per 4 μL.

[0055] II. Experimental process

[0056] (1) Prepare qPCR reaction solution according to the following table, and add 2 kinds of series RNA solution as template to each tube (only detect the corresponding fusion form for each RNA).

[0057]

[0058]

[0059] (2) Cover the tube cap, mix well by inverting, avoid air bubbles, and then centrifuge quickly.

[0060] (3) Run qPCR reaction according to the following table.

[0061]

[0062] III. Detection results

[0063] (1) Data list: see the following table for detection Ct value.

[0064] Fluorescence channel FAM HEX Cy5 Corresponding gene ALK ROS1 Housekeeping gene H2228-40ng 25.54 / 23.01 H2228-10ng 27.49 / 25.26 H2228-2.5ng 30.07 / 27.37 H2228-0.63ng 32.11 / 29.6 H2228-0.16ng 34.83 31.76 H2228-0.04ng 37.88 / 33.61 HCC78-40ng / 20.92 22.92 HCC78-10ng / 23.14 25.03 HCC78-2.5ng / 25.33 27.18 HCC78-0.63ng / 27.5 29.21 HCC78-0.16ng 29.59 31.47 HCC78-0.04ng / 31.62 35.61

[0065] (2) Data chart see Figure 1 and Figure 2 .

[0066] Four, result description

[0067] (1) The gradient dilution solution of two positive samples has good effect whether detecting corresponding fusion mutation or housekeeping gene, and Ct value and amplification spectrum show obvious gradient relationship.

[0068] (2) The detection reagent can detect fusion mutation in 0.04 ng positive sample.

[0069] Example three:

[0070] I. Sample preparation

[0071] 5 clinical FFPE samples were extracted for RNA, and Qubit was quantified.

[0072] II. Experimental process

[0073] (1) Prepare qPCR reaction solution according to the table below, and add 50 ng of clinical sample to each tube respectively.

[0074]

[0075] (2) Cover the tube cap, mix well by inverting, avoid air bubbles, and then centrifuge quickly.

[0076] (3) Run qPCR reaction according to the table below.

[0077]

[0078] III. Detection results

[0079] (1) The Ct value of tube 1 detection is shown in the table below.

[0080] Fluorescence channel FAM HEX Texas Red Cy5 Corresponding gene ALK NTRK1 NRG Housekeeping gene SMH 28.34 U.D U.D 26.14 WW U.D U.D 25.47 19.23 YZG U.D U.D U.D 29.17 WBJ U.D U.D 27.17 20.53 YYH U.D U.D U.D 29.69

[0081] (2) The Ct value of tube 2 detection is shown in the table below.

[0082] Fluorescence channel FAM HEX Cy5 Corresponding gene RET ROS1 Housekeeping gene SMH U.D U.D 23.45 WW U.D U.D 17.89 YZG U.D U.D 20.04 WBJ U.D U.D 27.64 YYH U.D U.D 23.83

[0083] IV. Result description

[0084] (1) The housekeeping gene of all samples was detected, and the sample quality was not a problem;

[0085] (2) SMH is ALK fusion, WW and WBJ are NRG fusion, and YZG and YYH are not detected fusion.

[0086] The primer composition of the present application can specifically amplify at least 50 fusion mutation forms of ALK, NRG, NTRK1, RET, ROS1 and the like 5 kinds of fusion driver genes in the RNA sample, and the housekeeping gene as the sample quality control. The reaction can be carried out in one tube, or can be carried out in multiple tubes. In the multiple tube reaction, a specific probe composition modified with a fluorescent group, such as the sequence shown in SEQ ID NO 55-70, can be added to form a qPCR reagent. When the qPCR reagent is used to detect positive samples, the fusion gene in as low as 0.04 ng of RNA can be reported. The process of the present application is simple, efficient, easy to operate, low in cost, and has good prospects.

[0087] The primer composition described in the present application comprises 54 primers, and the sequences are shown in SEQ ID 1-54. The first to 52th primers can match the breakpoint and surrounding sequence of ALK, NRG, NTRK1, RET, ROS1 and the like fusion driver genes, or CD74, EML4, KIF5B, SLC34A2, TPM3 and the like fusion partner genes, respectively. The 53th to 54th primers can specifically amplify the housekeeping gene in the sample.

[0088] The primer composition can specifically amplify at least 50 fusion forms of transcripts in the RNA sample by the method of multiplex PCR, and at the same time, the quality monitoring effect of the RNA sample can be achieved by amplifying the housekeeping gene. The primer composition can be subjected to multiplex PCR amplification in the same tube, or can be subjected to tube separation according to the needs, so as to meet the needs of different detection methods.

[0089] The reagent for fusion gene detection comprising the probe composition, the DNA sequence of the probe is shown in SEQ ID 55-70, and can be combined with one or more amplification products of the fusion gene. After the two ends of the probe are connected with different fluorescent reporter groups and / or fluorescent quenching groups, the above-mentioned primer composition can be used to report the fusion gene in the sample by the preferred reaction program and the method of multiplex qPCR.

[0090] The above-mentioned is only an embodiment of the present application, and does not limit the patent scope of the present application, and any equivalent structure or equivalent process transformation using the content of the present application specification, or direct or indirect application in other related technical fields, are also included in the patent protection scope of the present application.

[0091] Sequence listing:

[0092]

[0093]

[0094] sequence list <110> Shanghai Zhengu Biotechnology Co., Ltd. <120> A primer composition and a reagent for detecting fusion genes. <160> 70 <170> SIPOSequenceListing 1.0 <210> 1 <211> 30 <212> DNA <213> Human source <400> 1 ctgatgtttt gaggcgtctt gcaatctctg 30 <210> 2 <211> 30 <212> DNA <213> Human source <400> 2 acaagcataa agatgtcatc atcaaccaag 30 <210> 3 <211> 30 <212> DNA <213> Human source <400> 3 caaaactact gtagagccca cacctgggaa 30 <210> 4 <211> 30 <212> DNA <213> Human source <400> 4 tgttattaac tggaggaggg aaagacagaa 30 <210> 5 <211> 30 <212> DNA <213> Human source <400> 5 tatccacaca gacgggaatg aacagctctc 30 <210> 6 <211> 30 <212> DNA <213> Human source <400> 6 caccttgact ggtccccaga caacaagtat 30 <210> 7 <211> 30 <212> DNA <213> Human <400> 7 gactggtccc cagacaacaa gtatataatg 30 <210> 8 <211> 30 <212> DNA <213> Human <400> 8 tgcggagctt gctcagcttg tactcagggc 30 <210> 9 <211> 30 <212> DNA <213> Human <400> 9 gagcttgctc agcttgtact cagggctctg 30 <210> 10 <211> 30 <212> DNA <213> Human <400> 10 gaggtcttgc cagcaaagca gtagttgggg 30 <210> 11 <211> 30 <212> DNA <213> Human <400> 11 gggcagaggt caccacagag aggatcagcg 30 <210> 12 <211> 30 <212> DNA <213> Human <400> 12 ctgcaggtgg gtggtcagct gcaacatggc 30 <210> 13 <211> 30 <212> DNA <213> Human <400> 13 acgagcagct gagatgatgg catctttact 30 <210> 14 <211> 30 <212> DNA <213> Human <400> 14 gaaagcacac aaactgagag caacaaaaaa 30 <210> 15 <211> 30 <212> DNA <213> Human <400> 15 gaaaagacag ttggaggaat ctgtcgatgc 30 <210> 16 <211> 30 <212> DNA <213> Human <400> 16 ctttacacaa cctgcgcaaa ctctttgttc 30 <210> 17 <211> 30 <212> DNA <213> Human <400> 17 taatcttgaa cagctcacta aagtgcacaa 30 <210> 18 <211> 30 <212> DNA <213> Human <400> 18 cgcataaagg aagcagtcag gtcaaagaat 30 <210> 19 <211> 30 <212> DNA <213> Human <400> 19<400> 19 ggatagcttg gaaccacctg gagaaccagg 30 <210> 20 <211> 30 <212> DNA <213> Human origin <400> 20 ctcaggttat ggcagcaagt atgtctgctt 30 <210> 21 <211> 30 <212> DNA <213> Human origin <400> 21 cttgttgatt ccatagttcg tcagcgtgat 30 <210> 22 <211> 30 <212> DNA <213> Human origin <400> 22 gtggcctcaa ccatttccgg caaatcacag 30 <210> 23 <211> 30 <212> DNA <213> Human origin <400> 23 gtcaaaaact gggagagctt cggaaaaagc 30 <210> 24 <211> 30 <212> DNA <213> Human origin <400> 24 tcactcgtgc acatgaaagg gagtttggtt 30 <210> 25 <211> 30 <212> DNA <213> Human origin <400> 25 gaaatacggg acagaattga atcagggaga 30 <210> 26 <211> 30 <212> DNA <213> Human <400> 26 atcgcccccg tccacctgtc ttctgaagat 30 <210> 27 <211> 30 <212> DNA <213> Human <400> 27 gcagaagtgc ctggagaatg cccatctggc 30 <210> 28 <211> 30 <212> DNA <213> Human <400> 28 gtgacatcag caggttgaag gagcagctca 30 <210> 29 <211> 30 <212> DNA <213> Human <400> 29 cccacagcca ccgagacccc aaaaggtgtt 30 <210> 30 <211> 30 <212> DNA <213> Human <400> 30 ggacagggag ctgccaccca atgtcatgaa 30 <210> 31 <211> 30 <212> DNA <213> Human <400> 31 tcccccagct cccacttgag cacgatgtcc 30 <210> 32 <211> 30 <212> DNA <213> Human <400> 32 atgaagcact cccctccatt cacacagaaa 30 <210> 33 <211> 30 <212> DNA <213> Human origin <400> 33 caaagaaggc agagagagat gatttttgga 30 <210> 34 <211> 30 <212> DNA <213> Human origin <400> 34 gcactccttg gagcaaaagc ccactgacgc 30 <210> 35 <211> 30 <212> DNA <213> Human origin <400> 35 gtgagtcact ggaactggag gacccgtctt 30 <210> 36 <211> 30 <212> DNA <213> Human origin <400> 36 aagtgcaagg cactgcagga ggagaaccgc 30 <210> 37 <211> 30 <212> DNA <213> Human origin <400> 37 ggcctcacag atccagaagg caccagacgt 30 <210> 38 <211> 30 <212> DNA <213> Human origin <400> 38 gacccgtgct gagtttgctg agagatcggt 30 <210> 39 <211> 30 <212> DNA <213> Human <400> 39 gtcacatctt caggtgctgg atttttctta 30 <210> 40 <211> 30 <212> DNA <213> Human <400> 40 ccaggctgaa gtatatgggg cgagactagc 30 <210> 41 <211> 30 <212> DNA <213> Human <400> 41 gaaggaggtg gtaaccctgg tgctagttgc 30 <210> 42 <211> 30 <212> DNA <213> Human <400> 42 actttgagct gtctggctct ggagatctgg 30 <210> 43 <211> 30 <212> DNA <213> Human <400> 43 acatctttga gagaacggag gtcctggcag 30 <210> 44 <211> 30 <212> DNA <213> Human <400> 44 ttcttagtag cgccttccag ctggttggag 30 <210> 45 <211> 30 <212> DNA <213> Human <400> 45 aaggctcctg agacctttga taacataacc 30 <210> 46 <211> 30 <212> DNA <213> Human origin <400> 46 cttcagcttt ctcccactgt attgaatttt 30 <210> 47 <211> 30 <212> DNA <213> Human origin <400> 47 cctcgcagct cagccaactc tttgtcttcg 30 <210> 48 <211> 30 <212> DNA <213> Human origin <400> 48 caaaggctcc acttcccagc aagagacgca 30 <210> 49 <211> 30 <212> DNA <213> Human origin <400> 49 caggccgcca cactcctcac actccagccg 30 <210> 50 <211> 30 <212> DNA <213> Human origin <400> 50 gcagcaccga gacgatgaag gagaagagga 30 <210> 51 <211> 30 <212> DNA <213> Human origin <400> 51 cgggcaccgg aagaggagta gctgaccggg 30 <210> 52 <211> 30 <212> DNA <213> Human origin <400> 52 caccgtggtg taccctgctc tgcctttcag 30 <210> 53 <211> 30 <212> DNA <213> Human origin <400> 53 ccgggcacca ctccactgta tccctccccc 30 <210> 54 <211> 30 <212> DNA <213> Human origin <400> 54 ctttaggtca agtttacaac caagattcac 30 <210> 55 <211> 35 <212> DNA <213> Human origin <400> 55 tgtaccgccg gaagcaccag gagctgcaag ccatg 35 <210> 56 <211> 35 <212> DNA <213> Human origin <400> 56 aagtggcctg tgtagtgctt caagggccag gctgc 35 <210> 57 <211> 35 <212> DNA <213> Human origin <400> 57 tgtcacccac cccggagcca cacctgccac tctcg 35 <210> 58 <211> 35 <212> DNA <213> Human origin <400> 58 ctgtgggatc atgatctgaa tcctgaaaga gaaat 35 <210> 59 <211> 35 <212> DNA <213> Human origin <400> 59 acactaacag cacatctgga gacccggtgg agaag 35 <210> 60 <211> 35 <212> DNA <213> Human origin <400> 60 tgttcaccac atcaagcgcc gggacatcgt gctca 35 <210> 61 <211> 35 <212> DNA <213> Human origin <400> 61 cccggctgtg ctggctccag aggatgggct ggcca 35 <210> 62 <211> 35 <212> DNA <213> Human origin <400> 62 atctacatct acatccacca ctgggacaag ccatc 35 <210> 63 <211> 35 <212> DNA <213> Human origin <400> 63 tttgcccaca agccacccat ctcctcagct gagat 35 <210> 64 <211> 35 <212> DNA <213> Human origin <400> 64 CGACGAGCTG TGCCTACAGG TGATCGGAGC CGCTG 35 <210> 65 <211> 35 <212> DNA <213> Human <400> 65 ATCCAAAGTG GGAATTCCCT CGGAAGAACT TGGT 35 <210> 66 <211> 35 <212> DNA <213> Human <400> 66 CCCCCTGTCC TGTGCAGTCA GCAAGAGACG GCTGG 35 <210> 67 <211> 35 <212> DNA <213> Human <400> 67 CTGGAGTCCC AAATAAACCA GGCATTCCCA AATT 35 <210> 68 <211> 35 <212> DNA <213> Human <400> 68 CGTTTATAAG CACTGTCACC CCTTCCTTGG CACTT 35 <210> 69 <211> 35 <212> DNA <213> Human <400> 69 CCTGCCTTCC CTCGGGAAAA ACTGACTCTG CGTCT 35 <210> 70 <211> 35 <212> DNA <213> Human <400> 70 CTGGGATTGT ACCGCAGCTG CAAAATATTG TATCC 35

Claims

1. A primer composition for simultaneously detecting five lung cancer-related fusion genes, characterized in that, The primer composition consists of the nucleotide sequences shown in SEQ ID NO:1 to SEQ ID NO:54, and the primer composition is capable of specifically amplifying fusion gene fragments and housekeeping gene fragments from the ALK, ROS1, RET, NTRK1 and NRG1 genes in a multiplex PCR reaction system.

2. A reagent for detecting fusion genes, characterized in that, Includes the primer composition of claim 1.

3. The reagent for detecting fusion genes according to claim 2, characterized in that, The fusion form may be some or all of the following fusion forms: First fusion form: 5' end gene: EML4 e13, 3' end gene: ALK e20; Second fusion form: 5' end gene: EML4 e6, 3' end gene: ALK e20; Third fusion form: 5' end gene: EML4 e20, 3' end gene: ALK e20; Fourth fusion form: 5' end gene: EML4 e2, 3' end gene: ALK e20; Fifth fusion form: 5' end gene: EML4 e14, 3' end gene: ALK e20; Sixth fusion form: 5' end gene: EML4 e15, 3' end gene: ALK e20; Seventh fusion form: 5' end gene: EML4 e18, 3' end gene: ALK e20; Eighth fusion form: 5' end gene: EML4 e6, 3' end gene: ALK e19; Ninth fusion form: 5' end gene: KIF5B e15, 3' end gene: ALK e20; Tenth fusion form: 5' end gene: KIF5B e17, 3' end gene: ALK e20; Eleventh fusion form: 5' end gene: KIF5B e24, 3' end gene: ALK e20; The twelfth fusion form: 5' end gene: TFG e4, 3' end gene: ALK e20; Thirteenth fusion form: 5' end gene: TFG e5, 3' end gene: ALK e20; Fourteenth fusion form: 5' end gene: TPR e15, 3' end gene: ALK e20; The fifteenth fusion form: 5' end gene: HIP1 e28, 3' end gene: ALK e20; Sixteenth fusion form: 5' end gene: HIP1 e30, 3' end gene: ALK e20; Seventeenth fusion form: 5' end gene: KLC1 e9, 3' end gene: ALK e20; Eighteenth fusion form: 5' end gene: STRN e3, 3' end gene: ALK e20; Nineteenth fusion form: 5' end gene: CD74 e18, 3' end gene: NTRK1 e10; 20th fusion form: 5' end gene: TPM3 e8, 3' end gene: NTRK1 e10; 21st fusion form: 5' end gene: TFG e5, 3' end gene: NTRK1 e10; 22nd fusion form: 5' end gene: MPRIP e14, 3' end gene: NTRK1 e12; 23rd fusion form: 5' end gene: MPRIP e18, 3' end gene: NTRK1 e12; 24th fusion form: 5' end gene: MPRIP e21, 3' end gene: NTRK1 e12; 25th fusion form: 5' end gene: CD74 e8, 3' end gene: NRG1 e6; 26th fusion form: 5' end gene: CD74 e6, 3' end gene: NRG1 e6; 27th fusion form: 5' end gene: CD74 e6, 3' end gene: ROS1 e32; 28th fusion form: 5' end gene: CD74 e6, 3' end gene: ROS1 e34; 29th fusion form: 5' end gene: SDC4 e2, 3' end gene: ROS1 e32; Thirtieth fusion form: 5' end gene: SDC4 e2, 3' end gene: ROS1 e34; Thirty-first fusion form: 5' end gene: SDC4 e4, 3' end gene: ROS1 e32; Thirty-second fusion form: 5' end gene: SDC4 e4, 3' end gene: ROS1 e34; Thirty-third fusion form: 5' end gene: SLC34A2 e4, 3' end gene: ROS1 e32; Thirty-fourth fusion form: 5' end gene: SLC34A2 e4, 3' end gene: ROS1 e34; Thirty-fifth fusion form: 5' end gene: SLC34A2 e13, 3' end gene: ROS1 e32; Thirty-sixth fusion form: 5' end gene: SLC34A2 e13, 3' end gene: ROS1 e34; Thirty-seventh fusion form: 5' end gene: EZR e10, 3' end gene: ROS1 e34; Thirty-eighth fusion form: 5' end gene: TPM3 e8, 3' end gene: ROS1 e35; Thirty-ninth fusion form: 5' end gene: LRIG3 e16, 3' end gene: ROS1 e35; Fortieth fusion form: 5' end gene: GOPC e8, 3' end gene: ROS1 e35; 41st fusion form: 5' end gene: GOPC e4, 3' end gene: ROS1 e36; 42nd fusion form: 5' end gene: KIF5B e15, 3' end gene: RET e12; 43rd fusion form: 5' end gene: KIF5B e16, 3' end gene: RET e12; Forty-fourth fusion form: 5' end gene: KIF5B e22, 3' end gene: RET e12; 45th fusion form: 5' end gene: KIF5B e23, 3' end gene: RET e12; Forty-sixth fusion form: 5' end gene: KIF5B e15, 3' end gene: RET e11; 47th fusion form: 5' end gene: KIF5B e24, 3' end gene: RET e11; 48th fusion form: 5' end gene: KIF5B e24, 3' end gene: RET e8; 49th fusion form: 5' end gene: CCDC6 e1, 3' end gene: RET e12; The fiftieth fusion form: 5' end gene: CUX1 e10, 3' end gene: RET e12.

4. The reagent for detecting fusion genes according to claim 2, characterized in that, The reagent for detecting fusion genes also includes a probe composition for detecting fusion gene amplification products, wherein the probe sequence in the probe composition consists of the sequences shown in SEQ ID NO. 55~70.

5. The reagent for detecting fusion genes according to claim 4, characterized in that, In the probe composition, the 5' end of the probe can be connected to a fluorescent reporter group, and the 3' end of the probe can be selectively connected to a fluorescent quencher group.

6. The reagent for detecting fusion genes according to claim 2, characterized in that, The use of the reagents for fusion gene detection in the detection of fusion genes from RNA samples from fresh or frozen tissues, whole blood, and FFPE; the use of the reagents for fusion gene detection in the detection of fusion genes based on qPCR or NGS methods.

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