TW202122590A - Kit and methods to detect ntrk gene fusion - Google Patents

Abstract

Provided is a kit for detecting neurotrophin receptor tyrosine kinase (NTRK) gene fusion. The kit includes a set of NTRK fusion-specific primer pairs and a set of NTRK fusion-specific probes. Also provided is a method for detecting NTRK gene fusion, including generating an amplified target cDNA to hybridize with the set of NTRK fusion-specific probes in a single reaction, and detecting the probe-bound product to identify all possible NTRK gene fusions in a biological sample.

Description

Kit and method for detecting NTRK gene fusion

This application claims the priority of U.S. Provisional Application No. 62/893,148 filed on August 28, 2019, the entire content of which is incorporated herein by reference.

The present invention relates to kits and methods for molecular diagnosis and genomics. Specifically, the present invention relates to a kit and method for molecular diagnosis and cancer genomics.

The neurotrophin receptor tyrosine kinase (NTRK) gene family includes NTRK1, NTRK2, and NTRK3 genes, which encode three tropomyosin receptor kinases named TrkA, TrkB, and TrkC, respectively. ). When Trk receptors are activated, they trigger signaling pathways such as PI3K/AKT and MAPK, and regulate cell survival, proliferation, migration, differentiation, and synapse formation and plasticity. Trk receptors are expressed in many tissues, including nerve tissue, esophagus, stomach, breast, cervix, thyroid, lung, pancreas, colon, ovary and skin. Genome mutations involving NTRK, such as gene fusion, may cause a variety of adult and child tumors. A common type of gene fusion involved in NTRK gene is the fusion between the kinase domain of NTRK gene and a highly expressed 5'partner (5' partner), which results in abnormally high expression of Trk kinase domain. And activation. Since such gene variants are related to the fusion between a part of the NTRK gene and multiple possible fusion partners, there is a need for detection of multiple fusion types.

NTRK gene fusion can be used as a target for cancer therapy using TRK inhibitors such as larotrectinib and entrectinib. The mode of action of these drugs is to inhibit the kinase activity of the Trk receptor, thereby reducing downstream signals that promote cell proliferation. Although TRK inhibitors are effective for cancer patients involving NTRK gene fusion, only a small percentage of cancer patients have such genetic variants. Therefore, it is important to detect NTRK gene fusion before administering treatment, and to identify patients who may benefit from TRK inhibitor therapy.

Several techniques can be used to detect NTRK gene fusion, including next-generation sequencing (NGS), immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and Reverse transcription polymerase chain reaction (RT-PCR). Although next-generation sequencing can provide detailed and comprehensive information, it is costly and time-consuming, and requires more samples, which limits its clinical application. Immunohistochemical staining can detect the presence of NTRK protein, but it is difficult to distinguish the protein from NTRK fusion gene and wild-type NTRK gene. Fluorescence in situ hybridization can detect gene fusion, but it requires individual reactions to detect each type of fusion, and it requires well-trained experts to analyze the results. RT-PCR also requires different probes and individual reactions to detect each fusion type, so that as the number of fusion types to be detected increases, there is a greater demand for samples. Since more than one hundred fusion types of NTRK genes have been discovered so far, there is a need for novel detection methods that can detect multiple fusion types in a single reaction.

This disclosure relates to a method for detecting NTRK gene fusion. The method includes the following steps: (a) obtain ribonucleic acid (RNA) from a biological sample; (b) perform reverse transcription on the RNA to obtain complementary deoxyribonucleic acid (cDNA); (c) use at least two NTRK fusions NTRK fusion-specific primer pairs amplify the cDNA to obtain an amplified product; (d) mix the amplified product with at least two probes to obtain a probe-bound product ), wherein each of the probes has a different nucleotide sequence selected from the group consisting of SEQ ID NO: 1-125 and any complementary sequence thereof; and (e) detecting the probe binding product to determine the NTRK gene fusion exist.

The method disclosed in this article uses a set of specially designed probes, each probe can capture the amplification product containing a specific NTRK fusion sequence to detect all possible NTRK gene fusions. Since there are many types of NTRK gene fusions, but the exact one or more NTRK fusions in the biological sample cannot be known before the detection step, it is very important to obtain a detectable quantity of amplification products for all NTRK fusion types. In this way, any The NTRK fusion type can be detected later. Considering that different NTRK fusion-specific primer pairs show different amplification efficiencies, a pair of universal primers can be used in the previous step (c) to perform another DNA amplification to ensure that sufficient NTRK fusions are produced. Type of amplification product. Therefore, in some preferred embodiments, in step (c), at least two NTRK fusion-specific primer pairs are first used and then a universal primer pair is used to amplify the cDNA to obtain the amplified product . In this case, an NTRK fusion specific forward primer in each NTRK fusion specific primer pair further has the nucleotide sequence of a universal forward primer in the universal primer pair, and each NTRK fusion specific primer An NTRK fusion specific reverse primer in the pair further has the nucleotide sequence of a universal reverse primer in the universal primer pair.

On the other hand, this article also provides a kit for detecting NTRK gene fusion based on the aforementioned method. The set includes at least two NTRK fusion-specific primer pairs; and at least two probes, each probe having a different nucleotide selected from the group consisting of SEQ ID NO: 1-125 and any complementary sequence thereof sequence.

In some preferred embodiments, the set further includes a universal primer pair for another DNA amplification. In this case, an NTRK fusion specific forward primer in each NTRK fusion specific primer pair further has the nucleotide sequence of a universal forward primer in the universal primer pair, and each NTRK fusion specific primer An NTRK fusion specific reverse primer in the pair further has the nucleotide sequence of a universal reverse primer in the universal primer pair.

In some embodiments, the kit further includes a reverse transcriptase (reverse transcriptase) for reverse transcription of RNA isolated from biological samples, and a DNA polymerase (DNA polymerase) for amplification Increase the cDNA produced by reverse transcription.

The probe set composed of multiple probes used in the aforementioned method and set can target different NTRK fusions as targets with a high degree of specificity, and show similar hybrid efficiency to each other, thus ensuring accurate detection in a single reaction All possible NTRK gene fusions. Therefore, applying this method and set to NTRK fusion detection can reduce the need for large sample sizes and save detection time because there is no need to detect different NTRK fusion types separately. In addition, the method and kit are compatible with multiple platforms designed for multiplex reactions.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this disclosure belongs. definition

Unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" used in this article include plural referents.

The term "gene fusion" refers to the fusion of a first gene on a chromosome with a second gene on the same or different chromosome, resulting in the formation of a hybrid gene or a fusion gene . This phenomenon is also commonly referred to as "gene translocation" or "gene rearrangement". When the NTRK gene is one of multiple fused genes, such gene fusion is called "NTRK gene fusion" or "NTRK fusion". Usually, two "partner genes" are fused with each other. The gene located at the 5'end of the fusion gene is called "5' partner" or "5' gene", and the gene located at the 3'end of the fusion gene is called "3' partner" (3' partner)" or "3' gene (3' gene)". The fusion gene has a "fusion junction", which is the position where the 5'partner gene and the 3'partner gene are fused. The fusion junction is located in a fusion region defined by a fusion sequence (also called a fusion junction sequence), the fusion sequence having a sequence from the 5'gene and a sequence from the 3'gene. Different combinations of partner genes will lead to different "fusion types". Since the fusion junction may appear at any position within the partner gene, the fusion between two specific genes will further diverge due to the fusion junction. For example, the fusion between the first exon of the first gene and the second exon of the second gene is a type of fusion, while the third exon of the first gene and the first exon of the second gene Fusion between exons is another type of fusion.

Gene fusion can be detected by identifying a fusion junction located in a DNA or an RNA transcript of the DNA. The "fusion type" used herein refers to a specific fusion that exists in the RNA transcript. In other words, when the fusion lines between two specific genes occur at different sites in the same intron region, these fusions are regarded as the same type of fusion. For example, the fusion between exon 3 of gene A and exon 5 of gene B may have a DNA fusion region that contains a small part of the intron between exons 3 and 4 of gene A and Most of the introns between exons 4 and 5 of gene B. Alternatively, this fusion may have a DNA fusion region that contains most of the introns between exons 3 and 4 of gene A and a small part of the introns between exons 4 and 5 of gene B. section. Although the two fusions have different DNA fusion junctions, they are considered to be the same fusion type because the RNA transcripts produced by the two fusions are the same.

The term "primer" refers to a synthetic single-stranded oligonucleotide that can be used to amplify a target nucleic acid of a specific length. As used herein, the terms "NTRK fusion-specific primer" and "fusion specific primer" are used interchangeably, and refer to DNA primers designed to amplify a target cDNA And the target cDNA contains a fusion junction derived from a specific NTRK fusion gene. The NTRK fusion-specific primers are used in pairs, including an NTRK fusion-specific forward primer that can specifically bind to the 5'end of a target cDNA, and can specifically bind to the An NTRK fusion-specific reverse primer at the 3'end of the target cDNA.

The term "universal primer" as used herein refers to a DNA primer designed to amplify any DNA containing the nucleotide sequence of the universal primer. The universal primers are used in pairs and include a universal forward primer and a universal reverse primer.

Unless otherwise defined, the term "probe" or "NTRK fusion-specific probe" refers to a synthetic single-stranded DNA oligonucleotide that can be combined with a specific NTRK A fusion region of the fusion gene is heterozygous.

As used herein, "connector" or "linker" refers to a part of a molecule or a part of a complex composed of a plurality of molecules, which connects one molecule to another molecule. The linker or linker can function through covalent bonds, nucleic acid hybridization, or non-covalent interactions between a pair of molecules, such as biotin-streptavidin interactions. In this article, "connector" is used to connect a primer and a detectable molecule, such as a fluorescent molecule; "connector" is used to connect a probe and a detectable molecule or a unique recognition element ( unique identifier), such as a barcoded magnetic bead (BMB).

The present disclosure provides a method for detecting NTRK gene fusion. The method includes the following steps: (a) obtaining RNA from a biological sample; (b) performing reverse transcription on the RNA to obtain cDNA; (c) using at least two NTRK fusion-specific primer pairs to amplify the cDNA to obtain a Amplification product; (d) mixing the amplification product with at least two probes to obtain a probe binding product, each probe having a group selected from SEQ ID NO: 1-125 and any complementary sequence thereof Different nucleotide sequences of the group; and (e) detecting the probe binding product to determine the presence of gene fusion.

In step (a) of this method, RNA is prepared from a biological sample. The biological sample can be any sample obtained from an animal or a human subject. Examples of the biological sample include a formalin-fixed paraffin-embedded (FFPE) tissue section, blood, plasma or cells. In some embodiments, the biological sample is derived from a cancer patient. In some embodiments, the biological sample is derived from a solid tumor, soft tissue sarcoma, or hematological tumor. In some embodiments, the biological sample is derived from patients suffering from thyroid cancer, colorectal cancer, melanoma, soft tissue sarcoma, lung cancer, gastrointestinal stromal tumor, appendix cancer, breast cancer, salivary gland cancer, cholangiocarcinoma, pancreatic cancer, or A patient with infantile fibrosarcoma.

The preparation of total RNA from a biological sample can be performed by various methods known in the art. A typical procedure is to use organic solvents (such as phenol/chloroform) to extract RNA and precipitate it by centrifugation. Kits for isolating or purifying RNA are also available on the market. After RNA is obtained, the reverse transcriptase and four deoxyribonucleoside triphosphates (dNTPs, including dATP, dCTP, dTTP and dGTP) are used together to generate cDNA from the template RNA. This process is called Reverse Transcription. Reverse transcription can be performed using SuperScript cDNA synthesis kit (SuperScript cDNA synthesis kit; catalog number: 11754050, Invitrogen).

In step (c) of the method disclosed herein, DNA polymerase and at least two pairs of NTRK fusion-specific primers are used to amplify the cDNA to obtain an amplified product that will be used for probe detection. The amplification can be performed using a multiplex PCR kit containing DNA polymerase (product number: 206143, Qiagen). These NTRK fusion-specific primers can be provided as a reagent before use. In some embodiments, all NTRK fusion-specific primer pairs are pooled together to form a single pooled reagent. In other embodiments, the NTRK fusion-specific primer pairs are partially pooled to form a plurality of pooling reagents, and each reagent contains at least one NTRK fusion-specific primer pair. Therefore, the number of pooling reagents can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. In some preferred embodiments, more than one hundred NTRK fusion-specific primer pairs are provided in four pooling reagents for four multiplex amplification reactions. DNA amplification in this way has been confirmed to show significantly higher performance than using all NTRK fusion-specific primer pairs in a single multiplex amplification reaction, which may be due to the reduced primer complexity .

In some preferred embodiments, in step (c), at least two NTRK fusion-specific primer pairs are first used and then a universal primer pair is used to amplify the cDNA to obtain the amplified product. When the universal primer pair is used in the method disclosed herein, the NTRK fusion specific forward primer in each NTRK fusion specific primer pair further has the nucleotide sequence of the universal forward primer in the universal primer pair, And the NTRK fusion specific reverse primer in each NTRK fusion specific primer pair further has the nucleotide sequence of the universal reverse primer in the universal primer pair. The use of this universal primer pair can increase the final yield of any possible amplification product, no matter what NTRK fusion type will be detected or which NTRK fusion specific primer pair will be used in the first amplification. Another advantage of using a universal primer pair is that when you want to modify the primer to be detectable, you only need to modify two or even one universal primer, for example, between a universal primer and a linker (such as biotin). Bonding to connect a detectable molecule to the universal primer. Otherwise, all NTRK fusion-specific primer pairs must be modified, which will make the primer modification process more complicated and costly.

In step (d) of the method disclosed herein, the amplification product is mixed with at least two probes, so that a probe binding product can be formed by nucleic acid hybridization. Since these probes are specially designed based on the fusion sequence of each NTRK fusion type, by detecting specific probe binding products, the exact type of NTRK gene fusion can be determined. The NTRK gene fusions that can be detected include, but are not limited to, the fusion between the NTRK gene and a partner gene selected from the group consisting of: TFG (encoding trafficking from endoplasmic reticulum (ER) to Golgi regulator) ), ETV6 (encoding ETS variant transcription factor 6), QKI (encoding K-homology (KH) domain containing RNA binding protein), AFAP1 (encoding muscle Actin filament associated protein 1 (actin filament associated protein 1), BCAN (encoding brevican core protein), CD74 (encoding cluster of differentiation 74), CEL (encoding carboxyl ester Lipase (carboxyl ester lipase), CHTOP (encoding protein arginine methyltransferase 1 (PRMT1) chromatin target of PRMT1 protein), EPHB2 (encoding ephrin B receptor 2 ( ephrin type B receptor 2)), EPS15 (encoding epidermal growth factor receptor pathway substrate 15), IRF2BP2 (encoding interferon regulatory factor 2 binding protein 2) )), LMNA (encoding lamin A/C (lamin A/C)), MIR548F1 (encoding microRNA 548f-1 (microRNA 548f-1)), MPRIP (encoding myosin phosphatase Rho interacting protein ( myosin phosphatase Rho interacting protein), MRPL24 (encoding mitochondrial ribosomal protein L24), NFASC (encoding neurofascin), PEAR1 (encoding platelet endoth elial aggregation receptor 1)), PLEKHA6 (encoding pleckstrin homology domain containing A6), PPL (encoding periplakin), RNF213 (encoding ring finger protein 213 ( ring finger protein 213)), SQSTM1 (encoding selective sequestosome 1 (sequestosome 1)), SSBP2 (encoding single stranded DNA binding protein 2), TP53 (encoding tumor protein p53 (tumor protein p53)), TPM3 (encoding tropomyosin 3), TPM4 (encoding tropomyosin 4), TPR (encoding translocated promoter region, nuclear basket protein)), BCR (BCR activator of RhoGEF and GTPase), NACC2 (encoding NACC family member 2 (NACC family member 2)), PAN3 (encoding polyadenosine specific ribose) Nuclease subunit PAN3 (poly(A) specific ribonuclease subunit PAN3), STRN (coding for striatin), STRN3 (coding for striatin 3), TRIM24 (coding for triple motif protein 24 (tripartite motif) containing 24)), TRIM63 (encoding triple motif protein 63 (tripartite motif containing 63)), VCL (encoding vinculin), AKAP13 (encoding A-kinase anchoring protein 13), EML4 (encoding echinoderm microtubule binding protein 4 (EMAP like 4)), FAT1 (encoding FAT atypical cadherin 1), LYN (encoding a Src family tyrosine kinase (Src family tyrosine kinase) )), MYO5 A (encoding myosin VA (myosin VA)), RBPMS (encoding RNA binding protein, mRNA processing factor (RNA binding protein, mRNA processing factor)), VPS18 (encoding the CORVET complex and the VPS18 core subunit of the HOPS complex ( VPS18 core subunit of CORVET and HOPS complexes), DYNC2H1 (encoding dynein cytoplasmic 2 heavy chain 1), LAP3 (encoding leucine aminopeptidase 3), PDE4DIP (Encoding phosphodiesterase 4D interacting protein), GON4L (encoding gon-4 like protein), and CTRC (encoding chymotrypsin C). In a preferred embodiment, the detectable NTRK gene fusion types include TFG-NTRK1, ETV6-NTRK3, QKI-NTRK2, TPM3-NTRK1, ETV6-NTRK2, TFG-NTRK3, and NACC2-NTRK2. In another preferred embodiment, the detectable NTRK gene fusion types include PDE4DIP-NTRK1, TRIM63-NTRK1, GON4L-NTRK1 and CTRC-NTRK1.

Table 1 lists the probes specially designed to detect the NTRK gene fusion. Each of these probes has been proven to be specific to the type of gene fusion shown, and will not cross-react with other types of fusion, so the specific NTRK gene fusion type can be accurately determined. In some embodiments, in order to detect a specific NTRK gene fusion, a probe with any sequence of SEQ ID NO: 1-125 and a probe with a complementary sequence are used at the same time to enhance the detection efficiency. For example, in order to detect NTRK fusion 001 in Table 1, two probes with the sequence of SEQ ID NO: 1 and the sequence of SEQ ID NO: 126 can be used together. Table 1 NTRK Fusion Number 5 'gene (exon numbering) 3 'gene (exon numbering) Probe sequence ( from 5'end to 3'end) SEQ ID NO 001 TFG (5) NTRK1 (10) GTTAGTGTCTGAAACCT 1 AGGTTTCAGACACTAAC 126 CAGGTTTCAGACACTAACAG 2 CTGTTAGTGTCTGAAACCTG 127 002 ETV6 (5) NTRK3 (14) AATAGCAGGTCCCG 3 CGGGACCTGCTATT 128 GAATAGCAGGTCCCGTGG 4 CCACGGGACCTGCTATTC 129 003 ETV6 (4) NTRK3 (14) CGGGACCTTCTTCAT 5 ATGAAGAAGGTCCCG 130 CATGAAGAAGGTCCCGTGGC 6 GCCACGGGACCTTCTTCATG 131 004 QKI (6) NTRK2 (16) TGTATTAGGCCCAGC 7 GCTGGGCCTAATACA 132 TGGTGTATTAGGCCCAGCCTCC 8 GGAGGCTGGGCCTAATACACCA 133 005 TFG (6) NTRK1 (10) GTTAGTGTCTTCAATCTG 9 CAGATTGAAGACACTAAC 134 CAGATTGAAGACACTAACAG 10 CTGTTAGTGTCTTCAATCTG 135 006 AFAP1 (4) NTRK1 (10) ATCACATCAAACACTAACAG 11 CTGTTAGTGTTTGATGTGAT 136 007 BCAN (12) NTRK1 (10) GGGCTGGACACTAA 12 TTAGTGTCCAGCCC 137 008 BCAN (13) NTRK1 (11) AGAAGACCTGTCTCGGTG 13 CACCGAGACAGGTCTTCT 138 009 CD74 (7) NTRK1 (10) GGCCCAGACACTAA 14 TTAGTGTCTGGGCC 139 010 CD74 (8) NTRK1 (12) CCATGGGCCCGGC 15 GCCGGGCCCATGG 140 011 CEL (7) NTRK1 (7) CTGGAGTGTGTGCC 16 GGCACACACTCCAG 141 012 CHTOP (5) NTRK1 (10) GTGGTAGAGACACTAACA 17 TGTTAGTGTCTCTACCAC 142 013 CHTOP (5) NTRK1 (11) TGGTAGAGGTCTCGGTG 18 CACCGAGACCTCTACCA 143 014 EPHB2 (3) NTRK1 (9) GCCGAGTCCCGGC 19 GCCGGGACTCGGC 144 015 EPS15 (21) NTRK1 (9) CGATTCAGTCTCCTTCT 20 AGAAGGAGACTGAATCG 145 016 EPS15 (21) NTRK1 (9) CGATTCAGTCCCGGCC twenty one GGCCGGGACTGAATCG 146 017 IRF2BP2 (1) NTRK1 (10) ACTGCAGACACTAAC twenty two GTTAGTGTCTGCAGT 147 018 IRF2BP2 (1) NTRK1 (10) TCTAAAGCAGACACTAACAG twenty three CTGTTAGTGTCTGCTTTAGA 148 019 IRF2BP2 (1) NTRK1 (11) CTAAAGCAGGTCTCGGTG twenty four CACCGAGACCTGCTTTAG 149 020 IRF2BP2 (1) NTRK1 (9) CTAAAGCAGTCTCCTTCT 25 AGAAGGAGACTGCTTTAG 150 021 IRF2BP2 (1) NTRK1 (9) TAAAGCAGTCCCGGCCA 26 TGGCCGGGACTGCTTTA 151 022 LMNA (10) NTRK1 (11) CCACCACGTCTCGG 27 CCGAGACGTGGTGG 152 023 LMNA (10) NTRK1 (12) CACCACGCCCGGC 28 GCCGGGCGTGGTG 153 024 LMNA (10) NTRK1 (13) CCACCACGTGTTCA 29 TGAACACGTGGTGG 154 025 LMNA (11) NTRK1 (11) GCCCAGGTCTCGG 30 CCGAGACCTGGGC 155 026 LMNA (2) NTRK1 (10) GGCCAAGACACTAA 31 TTAGTGTCTTGGCC 156 027 LMNA (2) NTRK1 (11) GCCAAGGTCTCGG 32 CCGAGACCTTGGC 157 028 LMNA (3) NTRK1 (11) TACAGTGAGGTCTCGGTG 33 CACCGAGACCTCACTGTA 158 029 LMNA (4) NTRK1 (12) GCCAAGGCCCGGC 34 GCCGGGCCTTGGC 159 030 LMNA (5) NTRK1 (10) AGAAGCAGACACTAACA 35 TGTTAGTGTCTGCTTCT 160 031 LMNA (5) NTRK1 (11) AGAAGCAGGTCTCGGT 36 ACCGAGACCTGCTTCT 161 032 LMNA (6) NTRK1 (12) AGGAGAGGCCCGGC 37 GCCGGGCCTCTCCT 162 033 MIR548F1 (1) NTRK1 (10) AACCTAATAGACACTAACAGC 38 GCTGTTAGTGTCTATTAGGTT 163 034 MPRIP (14) NTRK1 (12) GAAGGAGGCCCGGC 39 GCCGGGCCTCCTTC 164 035 MPRIP (18) NTRK1 (12) CAACCAGGCCCGGC 40 GCCGGGCCTGGTTG 165 036 MPRIP (21) NTRK1 (12) GGATATGGCCCGGCT 41 AGCCGGGCCATATCC 166 037 MPRIP (21) NTRK1 (14) CGGATATGGCACTGAA 42 TTCAGTGCCATATCCG 167 038 MRPL24 (1) NTRK1 (9) AAAGTGGAGTCTCCTTCT 43 AGAAGGAGACTCCACTTT 168 039 MRPL24 (1) NTRK1 (9) AAGTGGAGTCCCGGCC 44 GGCCGGGACTCCACTT 169 040 NFASC (20) NTRK1 (10) GGAGAAGATTACACTAACAG 45 CTGTTAGTGTAATCTTCTCC 170 041 PEAR1 (15) NTRK1 (10) TCCCAGCACACTAA 46 TTAGTGTGCTGGGA 171 042 PEAR1 (2) NTRK1 (9) TGGGAAAGTCTCCTTCT 47 AGAAGGAGACTTTCCCA 172 043 PLEKHA6 (16) NTRK1 (10) TCAACAAAGACACTAACAG 48 CTGTTAGTGTCTTTGTTGA 173 044 PLEKHA6 (21) NTRK1 (9) CTGTCTGTCTCCTTC 49 GAAGGAGACAGACAG 174 045 PPL (12) NTRK1 (13) CTGACAGGTGTTCAC 50 GTGAACACCTGTCAG 175 046 PPL (22) NTRK1 (10) GTACGGTCACACTAAC 51 GTTAGTGTGACCGTAC 176 047 PPL (22) NTRK1 (11) TACGGTCGTCTCGGT 52 ACCGAGACGACCGTA 177 048 PPL (22) NTRK1 (13) TACGGTCGTGTTCAC 53 GTGAACACGACCGTA 178 049 RNF213 (15) NTRK1 (12) AAATTGAGGCCCGGCT 54 AGCCGGGCCTCAATTT 179 050 SQSTM1 (2) NTRK1 (10) TACATTAAAGACACTAACAGC 55 GCTGTTAGTGTCTTTAATGTA 180 051 SQSTM1 (5) NTRK1 (10) CCTCTGGACACTAAC 56 GTTAGTGTCCAGAGG 181 052 SQSTM1 (5) NTRK1 (12) CTCTGGGCCCGGC 57 GCCGGGCCCAGAG 182 053 SSBP2 (12) NTRK1 (12) CAGCAGGCCCGGC 58 GCCGGGCCTGCTG 183 054 TFG (4) NTRK1 (9) CTGAAAATGTCTCCTTCT 59 AGAAGGAGACATTTTCAG 184 CCTGAAAATGTCTCCTTCTC 60 GAGAAGGAGACATTTTCAGG 185 055 TFG (5) NTRK1 (9) GGTTTCAGTCCCGGCC 61 GGCCGGGACTGAAACC 186 CAGGTTTCAGTCCCGGCCAG 62 CTGGCCGGGACTGAAACCTG 187 056 TP53 (10) NTRK1 (9) ACTCCAGTCCCGGCC 63 GGCCGGGACTGGAGT 188 057 TP53 (11) NTRK1 (9) AGGGGTGTCCCGGC 64 GCCGGGACACCCCT 189 058 TP53 (8) NTRK1 (9) TAAGCGAGTCCCGGC 65 GCCGGGACTCGCTTA 190 059 TP53 (9) NTRK1 (9) CCTTCAGTCCCGGC 66 GCCGGGACTGAAGG 191 060 TPM3 (6) NTRK1 (11) TCAAGGAGGTCTCGGT 67 ACCGAGACCTCCTTGA 192 ACTCAAGGAGGTCTCGGTGG 68 CCACCGAGACCTCCTTGAGT 193 061 TPM3 (7) NTRK1 (12) TGGAAGGCCCGGC 69 GCCGGGCCTTCCA 194 ACCTGGAAGGCCCGGCTG 70 CAGCCGGGCCTTCCAGGT 195 062 TPM3 (7) NTRK1 (13) CTGGAAGGTGTTCA 71 TGAACACCTTCCAG 196 ACCTGGAAGGTGTTCACC 72 GGTGAACACCTTCCAGGT 197 063 TPM3 (7) NTRK1 (10) CCTGGAAGACACTAAC 73 GTTAGTGTCTTCCAGG 198 ACCTGGAAGACACTAACA 74 TGTTAGTGTCTTCCAGGT 199 064 TPM3 (7) NTRK1 (9) CTGGAAGTCTCCTT 75 AAGGAGACTTCCAG 200 GACCTGGAAGTCTCCTTCTC 76 GAGAAGGAGACTTCCAGGTC 201 065 TPM3 (8) NTRK1 (10) TTTTTTTAGAACACTAACAGC 77 GCTGTTAGTGTTCTAAAAAAA 202 AGGGTTTTTTTTAGAACACTAACAG 78 CTGTTAGTGTTCTAAAAAAAACCCT 203 066 TPR (10) NTRK1 (10) AAACGTAAAGACACTAACAG 79 CTGTTAGTGTCTTTACGTTT 204 067 TPR (16) NTRK1 (10) CCTTAAACAGACACTAACAG 80 CTGTTAGTGTCTGTTTAAGG 205 068 TPR (21) NTRK1 (10) ACTGGTAAAGACACTAACAG 81 CTGTTAGTGTCTTTACCAGT 206 069 TPR (21) NTRK1 (9) CTGGTAAAGTCTCCTTCT 82 AGAAGGAGACTTTACCAG 207 070 TPR (6) NTRK1 (12) AAGAAGAGGCCCGGCT 83 AGCCGGGCCTCTTCTT 208 071 AFAP1 (14) NTRK2 (12) GGTTCGAGTGCAAA 84 TTTGCACTCGAACC 209 072 BCR (1) NTRK2 (17) AGACGCAGTTGTTCAG 85 CTGAACAACTGCGTCT 210 073 ETV6 (4) NTRK2 (16) TGAAGAAGGCCCAGCC 86 GGCTGGGCCTTCTTCA 211 ACCATGAAGAAGGCCCAGCCTCC 87 GGAGGCTGGGCCTTCTTCATGGT 212 074 NACC2 (4) NTRK2 (13) GCTGTGAAATATTATGGAAC 88 GTTCCATAATATTTCACAGC 213 075 PAN3 (1) NTRK2 (17) GCTGGCAATTGTTCAG 89 CTGAACAATTGCCAGC 214 076 SQSTM1 (5) NTRK2 (13) CCCTCTGGATTATGGA 90 TCCATAATCCAGAGGG 215 077 SQSTM1 (5) NTRK2 (17) CCCTCTGGTTGTTCAG 91 CTGAACAACCAGAGGG 216 078 STRN3 (7) NTRK2 (16) GAATGGGGCCCAGC 92 GCTGGGCCCCATTC 217 079 STRN (3) NTRK2 (15) CTATGATTCTGATTTCTCATGG 93 CCATGAGAAATCAGAATCATAG 218 080 STRN (3) NTRK2 (16) TGATTCTGGCCCAGCC 94 GGCTGGGCCAGAATCA 219 081 TRIM24 (12) NTRK2 (15) CTTGCAGATTTCTC 95 GAGAAATCTGCAAG 220 082 TRIM24 (12) NTRK2 (16) CTTGCAGGCCCAGC 96 GCTGGGCCTGCAAG 221 083 VCL (16) NTRK2 (12) GACCCTGGTGCAAA 97 TTTGCACCAGGGTC 222 084 AKAP13 (14) NTRK3 (14) GATTGGACAGTCCCGTGG 98 CCACGGGACTGTCCAATC 223 085 AKAP13 (16) NTRK3 (15) AAGAGCAAAATGTGCAGC 99 GCTGCACATTTTGCTCTT 224 086 EML4 (6) NTRK3 (14) CAACCAAGGTCCCGTG 100 CACGGGACCTTGGTTG 225 087 EML4 (2) NTRK3 (14) AAGTAAAGGTCCCGTG 101 CACGGGACCTTTACTT 226 088 ETV6 (4) NTRK3 (15) ATGAAGAAGATGTGCAGC 102 GCTGCACATCTTCTTCAT 227 089 ETV6 (5) NTRK3 (15) GAATAGCAGATGTGCAGC 103 GCTGCACATCTGCTATTC 228 AGAATAGCAGATGTGCAGCA 104 TGCTGCACATCTGCTATTCT 229 090 FAT1 (2) NTRK3 (7) GAGACAGGCAGTTG 105 CAACTGCCTGTCTC 230 091 LYN (8) NTRK3 (14) TGGATGGGTCCCGT 106 ACGGGACCCATCCA 231 092 MYO5A (23) NTRK3 (11) ATGACAGAGAGCAC 107 GTGCTCTCTGTCAT 232 093 MYO5A (23) NTRK3 (12) AGATGACAGTTGACGAAG 108 CTTCGTCAACTGTCATCT 233 094 MYO5A (32) NTRK3 (12) GAACTAGAAGTTGACGAAGT 109 ACTTCGTCAACTTCTAGTTC 234 095 MYO5A (32) NTRK3 (13) GAACTAGAAGGTATCCATAG 110 CTATGGATACCTTCTAGTTC 235 096 RBPMS (5) NTRK3 (14) AGAGCCATGTCCCGTG 111 CACGGGACATGGCTCT 236 097 TFG (6) NTRK3 (10) AGATTGAAGATCCCCCAC 112 GTGGGGGATCTTCAATCT 237 CAGATTGAAGATCCCCCACG 113 CGTGGGGGATCTTCAATCTG 238 098 TFG (6) NTRK3 (14) AGATTGAAGGTCCCGTGG 114 CCACGGGACCTTCAATCT 239 099 TFG (6) NTRK3 (15) AGATTGAAGATGTGCAGC 115 GCTGCACATCTTCAATCT 240 100 TPM4 (7) NTRK3 (12) CCTGGAAGTTGACGAA 116 TTCGTCAACTTCCAGG 241 101 VPS18 (4) NTRK3 (18) CCTGCAGGTGGGAG 117 CTCCCACCTGCAGG 242 102 NTRK1 (16) DYNC2H1 (85) CACGGAGTGTCCTC 118 GAGGACACTCCGTG 243 103 NTRK1 (9) TFG (6) ATCCCTGGGCCACC 119 GGTGGCCCAGGGAT 244 104 NTRK1 (12) TPR (6) TGATGCCTTATCGAGA 120 TCTCGATAAGGCATCA 245 105 NTRK1 (9) TPR (22) ATCCCTGGTCAGCC 121 GGCTGACCAGGGAT 246 106 NTRK2 (13) LAP3 (1) TCATCTCCAAAAAA 122 TTTTTTGGAGATGA 247 107 NTRK3 (13) ETV6 (5) GAATGAAGGATAACTGTG 123 CACAGTTATCCTTCATTC 248 108 NTRK3 (13) RBPMS (6) AATGAAGGATGAGCTC 124 GAGCTCATCCTTCATT 249 109 NTRK3 (14) ETV6 (6) GGACACGTACTGTAGA 125 TCTACAGTACGTGTCC 250

In some embodiments, after a target cDNA is amplified and probed, an NTRK fusion type is detected. In other embodiments, two or more target cDNAs with different sequences are amplified in one reaction (referred to as multiple amplification reaction) and/or detected in one reaction (referred to as multiple hybrid reaction). ) After that, multiple NTRK fusion types can be detected at the same time. When the method disclosed herein is performed in a multiple reaction scenario, at least two probes for detecting at least two NTRK gene fusion types are used. In some embodiments, the at least two probes are selected from the group consisting of SEQ ID NO: 1-125 and any complementary sequence thereof. In some preferred embodiments, the at least two probes are selected from SEQ ID NO: 2, 4, 6, 8, 10, 60, 62, 68, 70, 72, 74, 76, 78, 87, 88 , A group consisting of 102, 104, 113, 114, 115 and any complementary sequence thereof. More preferably, the at least two probes are selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 62, 70, 87, 104, 113 and any complementary sequence thereof. These probes can be provided as a single pooling reagent or as separate multiple reagents.

Generally, probes and amplification products are mixed at a specific temperature to promote probe hybridization. The optimal thermal mixing conditions for probe hybridization will vary with the probe sequence. Therefore, for multiple reactions using at least two probes, it is very difficult to select a suitable hybridization condition for all probes. However, by using the probes listed in Table 1, multiple reactions can be performed at a fixed temperature with a fixed shaking speed, because these probes are designed to hybridize with their respective targets under similar hybrid conditions. . In some embodiments, the temperature used for hybridization is between 35-50°C, 40-50°C, 40-45°C, or 45-50°C. In some embodiments, the hybrid system is between 700-1000 rpm, 750-1000 rpm, 800-1000 rpm, 900-1000 rpm, 700-750 rpm, 700-800 rpm, 750-800 or 800-900 It is carried out by using a thermal mixer at a rotation speed of rpm.

The detection of probe binding products can be achieved by detecting NTRK fusion-specific primers, universal primers, or probes in the product. Therefore, primers or probes are usually modified to be detectable. By connecting them directly or indirectly to a detectable molecule, the primers or probes can be modified to have fluorescent or chemiluminescent activity, or become colorable or colorimetric. In some embodiments, one or two primers in the primer pair are linked to biotin or other compounds capable of binding to a streptavidin-conjugated detectable molecule. The detectable molecule can be a dye, a fluorescent molecule, such as phycoerythrin (PE) or cyanines, or an enzyme used in a color reaction, such as alkaline phosphoric acid Enzyme (alkaline phosphatase, AP) or horseradish peroxidase (horseradish peroxidase, HRP). The enzyme used in the color reaction catalyzes the production of colored compounds when a color substrate is present.

In some embodiments, the probe system for detecting a specific NTRK fusion type is connected to a unique identification element, so that multiple NTRK fusion types can be detected simultaneously and can be distinguished from each other. The unique identification element can be an oligonucleotide with a unique sequence, or it can be a microbead or nanoparticle containing a unique barcode on the surface. The barcode can be a geometric pattern that can be read by an optical scanner with a bright-field imaging system. In some embodiments, the microbead or the nanoparticle is a magnetic particle. In some embodiments, the microbeads or the nanoparticles are made of synthetic polymers.

The unique identification element can be connected to the probe directly or via a linker. In some embodiments, the unique identification element is connected to the probe by direct chemical coupling, so a covalent bond is formed between the unique identification element and the probe. In some embodiments, the unique identification element is connected to the probe through a polymer linker. In some embodiments, the unique recognition element is connected to the probe through hybridization between complementary nucleotide sequences.

The method disclosed herein can be executed on several technology platforms capable of performing multiple reactions, such as a microarray plate, a gene chip, microbeads, nanoparticles, a membrane or A microfluidic device. In some embodiments, the probes are fixed to different positions on a microarray plate, a gene chip or a membrane, for example, in the form of a dot array, and each dot contains a plurality of types of probes. In other embodiments, the probe system is connected to microbeads (for example, magnetic microbeads). In other embodiments, the probes are coated on a substrate of a microfluidic device, wherein different probes are located in different regions of the substrate.

When the probe is fixed on a DNA microarray plate, the microarray plate may further include a set of control points, and each control point includes a plurality of control probes. The control probe will interact with housekeeping genes (beta-actin), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and β2-microglobulin (beta-actin). 2-microglobulin)) cDNA binding. Therefore, the control point can be used as an internal control group to check the effectiveness of the test. In addition, the microarray plate may further include a set of anchor points, and each anchor point includes a plurality of anchor probes. The anchor probe is designed to be detected regardless of the amplified product. Therefore, the anchor point can be used as a position indicator of a point near the microarray plate.

This article also provides a kit for detecting NTRK gene fusion based on the method disclosed herein. The set includes at least two NTRK fusion-specific primer pairs; and at least two probes, each probe having a different nucleotide selected from the group consisting of SEQ ID NO: 1-125 and any complementary sequence thereof sequence.

In some preferred embodiments, each set further includes a universal primer pair. When the NTRK fusion specific primer pair is used in combination with a universal primer pair, the NTRK fusion specific forward primer in each NTRK fusion specific primer pair further has the nucleotide sequence of the universal forward primer in the universal primer pair And the NTRK fusion-specific reverse primer in each NTRK fusion-specific primer pair further has the nucleotide sequence of the universal reverse primer in the universal primer pair. In addition, each of the at least two probes has a difference that is preferably selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 62, 70, 87, 104, 113 and any complementary sequence thereof. Nucleotide sequence.

In some embodiments, the kit further includes a reverse transcriptase for reverse transcription of RNA isolated from a biological sample, and a DNA polymerase for amplifying cDNA generated by reverse transcription .

In some embodiments, the set further includes an internal control group. The internal control group can be a positive control sample with NTRK gene fusion, or can be a negative control sample without NTRK gene fusion. In some embodiments, the internal control group is a FFPE tissue section, blood, plasma, cells, nucleic acids or oligonucleotides.

In some embodiments, when one or more NTRK gene fusions are detected in a biological sample from a cancer patient, the patient is expected to respond to a TRK inhibitor, particularly an NTRK inhibitor, such as Larote Larotrectinib, entrectinib, LOXO-195 or TPX-0005. Examples Example 1 Detection of NTRK gene fusion with a single amplification target - probe- BMB hybrid test

A single amplification target-probe-barcode magnetic bead (BMB) hybrid test can simultaneously detect multiple possible NTRK fusions in a single reaction. Figure 1 shows the entire process of the experiment, which includes the following steps: obtain RNA from a biological sample, reverse transcription of the RNA to obtain cDNA, and use a variety of NTRK fusion-specific primer pairs to amplify by polymerase chain reaction (PCR) Increase the NTRK fusion region (ie target cDNA) of the cDNA to obtain an amplified product of the target cDNA, and use BMB-coupled probes to hybridize with the amplified target cDNA and detect probes. Needle binding product. The following is an example of this test. Preparation of NTRK Fusion Specific Probe

Before testing, based on the nucleotide sequence of the fusion region in the RNA transcript of each NTRK fusion gene (Table 2), five NTRK fusion specific probes were designed. The first 20 base pairs and the last 20 base pairs of all sequences in Table 2 are derived from the 5'partner gene and the 3'partner gene, respectively. In order to hybridize at the bottom line in Table 2, each probe was designed to have the sequence listed in Table 3. These probes were synthesized by IDT (Integrated DNA Technologies, Inc., Coralville, Iowa) and were given an amine modification at the 5'end. Subsequently, the five fusion-specific probes are coupled to five BMBs with different identification codes through an amine-carboxy bond, so that when different probes are used in a multiple reaction, each probe can be distinguished. Table 2 NTRK Fusion Number 5 'Gene 3 'Gene Fusion region sequence ( from 5'end to 3'end) SEQ ID NO T5N10 TFG NTRK1 TAACAGATGATC AGGTTTCA GACACTAAC AGCACATCTGG 251 E5N14 ETV6 NTRK3 TGCCCATTGGGAG AATAGCA GGTCCCG TGGCTGTCATCAG 252 E4N14 ETV6 NTRK3 TGCATCAGAACC ATGAAGAA GGTCCCG TGGCTGTCATCAG 253 Q6N16 QKI NTRK2 TTGAACCTAGTGG TGTATTA GGCCCAGC CTCCGTTATCAG 254 T6N10 TFG NTRK1 CTCAAGCAGGT CAGATTGAA GACACTAAC AGCACATCTGG 255 table 3 NTRK Fusion Number Probe sequence ( from 5'end to 3'end) BMB identification code T5N10 GTTAGTGTCTGAAACCT (SEQ ID NO:1) 40 E5N14 CGGGACCTGCTATT (SEQ ID NO: 128) 59 E4N14 CGGGACCTTCTTCAT (SEQ ID NO: 5) 52 Q6N16 GCTGGGCCTAATACA (SEQ ID NO: 132) 65 T6N10 GTTAGTGTCTTCAATCTG (SEQ ID NO: 9) 75 PCR amplification using NTRK fusion-specific primer pair

In order to replace clinical samples with NTRK gene fusion, five oligonucleotides (called NTRK fusion oligo) with the sequence of SEQ ID NO: 251-255 synthesized by IDT were used as a positive control Set of templates. The five NTRK fusion-specific primer pairs shown in Table 4 were used to amplify the five NTRK fusion oligomers by multiplex PCR. Each primer pair capable of binding the 5'end and 3'end of the respective NTRK fusion oligomers was synthesized by IDT. The 5'end of the reverse primer in each primer pair is modified by biotin to interact with the streptavidin-phycoerythrin (SA-PE) conjugate (Thermo Fisher Scientific). The PCR system used high-fidelity Platinum Taq DNA polymerase (Thermo Fisher Scientific) to perform 30 thermal cycles on a Veriti™ 96-well thermal cycler (Thermo Fisher Scientific) according to the manufacturer's instructions. Table 4 NTRK Fusion Number NTRK fusion sequence-specific primer pair (from 5 'to 3' end) SEQ ID NO T5N10 Positive ATGTCAGCGTTTGGCTT 256 Reverse TTTCGTCCTTCTTCTCCACC 257 E5N14 Positive CCACATCATGGTCTCTGTCT 258 Reverse GGCTGAGTCCTCCTCAC 259 E4N14 Positive CAGCCGGAGGTCATACT 260 Reverse GGCTGAGTCCTCCTCAC 261 Q6N16 Positive CCAGCTACATCAATCCTTGAG 262 Reverse CTGGCAGAGTCATCATCATT 263 T6N10 Positive ACAGCAGCCACCATATACA 264 Reverse AGGTGTTTCGTCCTTCTT 265 Probe hybridization and signal detection

The amplification product of each NTRK fusion oligomer was mixed with five probes-BMB in the same hole of a 96-well plate for hybridization. The hybrid system is performed at 40-45°C and shaking at about 700 rpm for 10-30 minutes. After hybridization, a fluorescent SA-PE conjugate is added to the hole to bind to the biotin of the amplified product, and the probe-BMB is washed to remove unbound materials. In addition, another BMB (identification code 0) that does not carry the probe is added to the hole to serve as a negative control group. Finally, a BioCode 2500 analyzer (Applied BioCode Inc., Taipei, Taiwan) equipped with a camera capable of performing bright-field and fluorescent imaging at the same time was used to read the BMB's barcode and detect the BMB's fluorescent signal.

Table 5 shows the fluorescence intensities of BMB59, BMB65, BMB40, BMB52, BMB75 and BMB0. According to Table 5, the fluorescence intensity of a specific BMB indicating a specific target-probe hybrid (that is, a specific NTRK fusion type is present) is significantly higher than that of a non-specific signal. This result indicates that a single amplification target-probe-BMB test can be used to detect and distinguish multiple types of NTRK gene fusions. table 5 BMB identification code oligomer number 0 59 65 40 52 75 SEQ ID NO: 252 7 741 7 - - - SEQ ID NO:254 4 4 1062 - - - SEQ ID NO: 251 7 - - 980 205 192 SEQ ID NO: 253 5 - - 152 556 116 SEQ ID NO: 255 5 - - 124 123 1959 Example 2 Detection of NTRK gene fusion by secondary amplification target - probe hybrid test

The secondary amplification target-probe hybrid test is another method designed to simultaneously detect multiple possible NTRK fusions in a single reaction. Figure 2 shows the entire process of the experiment, which includes the following steps: RNA is obtained from a biological sample, the RNA is reverse transcribed to obtain cDNA, and multiple NTRK fusion-specific primer pairs are used to amplify by polymerase chain reaction (PCR) The NTRK fusion region of the cDNA (ie the target cDNA) to obtain the first amplification product of the target cDNA, a universal primer pair is used to amplify the first amplification product by PCR to obtain the second amplification product of the target cDNA To increase the product, the amplified target cDNA is used for probe hybridization and detection of probe binding products. The following is an example of this test. RNA extraction and reverse transcription

The RecoverAll Total Nucleic Acid Separation Kit (Cat. No.: AM1975, Ambient Technologies) was used to extract DNA and RNA from FFPE tissue samples of cancer patients according to the manufacturer's instructions. Using SuperScript cDNA synthesis kit (Cat. No.: 11754050, Invitrogen) and random hexanucleotide primers (random hexanucleotide primers) were used to reverse transcribed 100 ng of total RNA at 42°C for 30 to 60 minutes. This step generates 10 μL of cDNA product. PCR amplification using NTRK fusion-specific primer pair

Each primer in the NTRK fusion-specific primer pair used in this experiment was designed to have two fragments. A fragment is called a fusion-specific fragment, which is used to bind the 5'end or 3'end of a fusion sequence of a specific NTRK fusion. The other fragment is called the universal fragment, which has the nucleotide sequence of the universal primer used in the second PCR. The universal fragment is always upstream or 5'position relative to the fusion-specific fragment (Figure 2). The universal primer can be any of the primers listed in Table 6, and each universal primer in Table 6 can be used as a universal forward primer or a universal reverse primer. Table 7 shows the fusion-specific fragments of some fusion-specific primer pairs used in this experiment. Table 6 Universal primer number Primer sequence ( 'end to the 3' end of 5) SEQ ID NO U01 GTTTTCCCAGTCACGACGT 266 U02 GCAAATGGCATTCTGACATCC 267 U03 GCGGATAACAATTTCACACAGG 268 U04 CGTCCATGCCGAGAGTG 269 U05 CTTTATGTTTTTGGCGTCTTCCA 270 U06 GACTGGTTCCAATTGACAAGC 271 U07 GCGTGAATGTAAGCGTGAC 272 U08 TGTAAAACGACGGCCAGT 273 U09 AAGGGTCTTGCGAAGGATAG 274 U10 GGGTTATGCTAGTTATTGCTCAG 275 Table 7 NTRK Fusion Number NTRK fusion sequence specific primers specific to the fusion fragment (from 5 'to 3' end) SEQ ID NO 001 Positive ATGTCAGCGTTTGGCTT 276 Reverse TTTCGTCCTTCTTCTCCACC 277 002 Positive CCACATCATGGTCTCTGTCT 278 Reverse GGCTGAGTCCTCCTCAC 279 003 Positive CAGCCGGAGGTCATACT 280 Reverse GGCTGAGTCCTCCTCAC 281 004 Positive CCAGCTACATCAATCCTTGAG 282 Reverse CTGGCAGAGTCATCATCATT 283 005 Positive ACAGCAGCCACCATATACA 284 Reverse AGGTGTTTCGTCCTTCTT 285 055 Positive TGGCTTAACAGATGATCAGG 286 Reverse GAGAAGGGGATGCACCA 287 061 Positive GACCCGTGCTGAGTTTG 288 Reverse CAGCCCATCCTCTGGAG 289 073 Positive GAGGTCATACTGCATCAGAAC 290 Reverse CATTGGAGATGTGATGGAGTG 291 089 Positive TCCCCGCCTGAAGAGCA 292 Reverse TCTCGCTTCAGCACGAT 293 097 Positive ACCATATACAGGAGCTCAGAC 294 Reverse CTCGATGCAGTGCTCCA 295

When performing fusion-specific PCR, add 7 μL of water to 10 μL of cDNA product, and then divide the resulting mixture (17 μL) into four groups of pools, each with 4 μL of the mixture, and the remaining 1 Dead volume of μL. The number of pools depends on the efficiency of the primer. More specifically, the primer efficiency of each fusion-specific primer pair is measured first, and then fusion-specific primer pairs with similar efficiencies are mixed to form a single primer pool, and finally a total of four primer pools (represented as P1, P2, P3, P4). Each primer pool containing 23 to 48 fusion-specific primers (Table 8) was added to a set of cDNA (4 μL). After that, according to the manufacturer's instructions, a multiplex PCR kit (Cat. No.: 206143, Qiagen) was used to amplify each set of cDNA on a Veriti™ 96-well thermal cycler (Thermo Fisher Scientific) for 25 thermal cycles, thereby generating 10 μL The first amplified product. In other words, four multiplex PCR reactions are performed to generate four sets of first amplification products. Table 8 Collection number Number of primers Number of fusion gene NTRK Number of housekeeping genes P1 48 39 1 P2 35 25 1 P3 43 28 1 P4 twenty three 16 0 PCR amplification using universal primer pairs

Since each fusion-specific primer contains the nucleotide sequence of a universal primer at the 5'end, the first amplification product can be further amplified by PCR using a universal primer pair. The universal primer pair includes a universal forward primer having a sequence selected from SEQ ID NO: 266-275, and a universal reverse primer having a sequence selected from SEQ ID NO: 266-275. The universal reverse primer is modified with biotin. When performing the second PCR, dilute each of the aforementioned four sets of first amplification products 100 times into the final reaction mixture, and use the Platinum SuperFi II PCR master mix (Cat No: 12368010, Invitrogen) in accordance with the manufacturer’s instructions. The first amplification product of each group was amplified for 25 thermal cycles on the Veriti™ 96-well thermal cycler (Thermo Fisher Scientific) to obtain 10 μL of the second amplification product. In other words, four PCR reactions are performed to generate four sets of second amplification products. Probe hybridization and signal detection

The aforementioned four sets of second amplification products were pooled (40 μL in total), and 18 μL of the resulting pool was mixed with 3 μL of water to produce a mixture. The mixture was placed in a 96-well PCR plate (Cat. No.: P46-4TI-1000/C, 4titude). The second amplified product was denatured at 96°C for 5 minutes, and then moved into pre-blocked wells. Each well was pre-printed with an array of probe spots, including NTRK fusion-specific probes. 117 points, 9 points for the control probe, and 10 points for the anchor probe. The NTRK fusion specific probe includes SEQ ID NOs: 2, 4, 6, 8, 10, 60, 62, 68, 70, 72, 74, 76, 78, 87, 88, 102, 104, 113, 114 , 115 sequence probe. Figure 3 shows the distribution of different probes in a hole. The target-probe hybrid system was shaken at 50°C for 15 minutes. After hybridization, the holes are cooled and cleaned twice. Subsequently, the buffer containing the streptavidin-alkaline phosphatase conjugate is added to the hole to allow the biotin-streptavidin to interact, and then the substrate for alkaline phosphatase is added so that the colored products can be probed. The needle-target hybrid is generated at the location. By taking a picture of the hole with a camera and identifying the position of the colored spot in the hole, it is possible to determine the specific heterozygosity indicating the presence of a specific NTRK fusion. The analysis of the position of the colored spots can be performed by a computer.

no.

Those skilled in the art will clearly understand the present invention by virtue of the following detailed description of the preferred embodiments and the accompanying drawings, in which:

Figure 1 is a schematic diagram of a NTRK gene fusion detection method according to an embodiment of the present invention; the detection is based on a single-amplified target-probe-barcoded hybrid test (BMB) magnetic bead (BMB) hybridization assay);

Figure 2 is a schematic diagram of an NTRK gene fusion detection method according to an embodiment of the present invention; the detection is based on a double-amplified target-probe hybridization assay; and

Figure 3 shows an array of probe points printed in a hole of a multi-well disk; the numbers in the first row of the top and the first column on the left are used as reference coordinates; the squares marked 1-117 indicate NTRK fusion specificity The points of the probe, the squares labeled IC001-IC009 represent the points of control probes, and the squares labeled R144 represent the points of anchor probes.

no.

Claims (21)

A kit for detecting neurotrophic receptor tyrosine kinase (NTRK) gene fusion, comprising: at least two NTRK fusion-specific primer pairs; and at least two probes, each of which has a probe selected from SEQ ID NO : 2, 4, 6, 8, 10, 62, 70, 87, 104, 113 and any of the complementary sequences of different nucleotide sequences in the group. The set according to claim 1, further comprising a universal primer pair, wherein one NTRK fusion specific forward primer in each NTRK fusion specific primer pair further has a universal primer pair in the universal primer pair Nucleotide sequence, and an NTRK fusion-specific reverse primer in each NTRK fusion-specific primer pair further has the nucleotide sequence of a universal reverse primer in the universal primer pair. The kit according to claim 1, further comprising an additional probe, the additional probe having one selected from SEQ ID NO: 1, 3, 5, 7, 9, 11-61, 63-69, 71-86 , 88-103, 105-112, 114-125 and any of its complementary sequences to form a group of nucleotide sequences. The kit according to claim 1, wherein one NTRK fusion specific forward primer or one NTRK fusion specific reverse primer in each NTRK fusion specific primer pair is combined with a detectable molecule. The kit according to claim 4, wherein the detectable molecule is a fluorescent molecule or an enzyme for a color reaction. The kit according to claim 2, wherein the universal forward primer or the universal reverse primer is combined with a detectable molecule. The kit according to claim 6, wherein the detectable molecule is a fluorescent molecule or an enzyme for a color reaction. The kit according to claim 1, wherein the at least two probes are fixed on different positions on a microarray plate. The kit according to claim 8, wherein the microarray plate includes a position indicator. The kit according to claim 1, further comprising a control probe for detecting a housekeeping gene. The kit according to claim 1, further comprising a reverse transcriptase and a DNA polymerase. A method for detecting neurotrophic receptor tyrosine kinase (NTRK) gene fusion, including the following steps: (a) Obtain ribonucleic acid (RNA) from a biological sample; (b) Reverse transcription of the RNA to obtain complementary deoxyribonucleic acid (cDNA); (c) Amplify the cDNA using at least two NTRK fusion-specific primer pairs to obtain an amplified product; (d) mix the amplified product with at least two probes to obtain a probe binding product, wherein each of the The probe has different nucleotide sequences selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 62, 70, 87, 104, 113 and any complementary sequence thereof; and (e) Detect the probe binding product to determine the presence of NTRK gene fusion. The method according to claim 12, wherein in step (c), the at least two NTRK fusion-specific primer pairs are first used and then a universal primer pair is used to amplify the cDNA to obtain the amplified product, wherein each An NTRK fusion specific forward primer in the NTRK fusion specific primer pair further has the nucleotide sequence of a universal forward primer in the universal primer pair, and one NTRK fusion specific primer in each NTRK fusion specific primer pair is fused The specific reverse primer further has the nucleotide sequence of a universal reverse primer in the universal primer pair. The method according to claim 12, wherein in step (d), the amplified product is further mixed with an additional probe, wherein the additional probe has one selected from SEQ ID NO: 1, 3, 5, 7, 9 , 11-61, 63-69, 71-86, 88-103, 105-112, 114-125 and any of the complementary sequences of the nucleotide sequence of the group. The method according to claim 12, wherein an NTRK fusion specific forward primer or an NTRK fusion specific reverse primer in each NTRK fusion specific primer pair is combined with a detectable molecule. The method according to claim 15, wherein the detectable molecule is a fluorescent molecule or an enzyme for a color reaction. The method according to claim 13, wherein the universal forward primer or the universal reverse primer is combined with a detectable molecule. The method according to claim 17, wherein the detectable molecule is a fluorescent molecule or an enzyme for a color reaction. The method according to claim 12, wherein in step (d), the at least two probes and the amplification product are mixed at a temperature between 35-50°C. The method according to claim 12, wherein in step (d), the at least two probes and the amplification product are mixed at a rotation speed of 700-1000 rpm. The method according to claim 12, wherein the at least two probes are fixed on different positions on a microarray plate.

Images