Primer probe combination for jointly detecting EML4-ALK fusion and ROS1 fusion in plasma exosome and application of primer probe combination

Abstract

The invention provides a primer probe combination for jointly detecting EML4-ALK fusion and ROS1 fusion in a plasma exosome and application of the primer probe combination. Detection sites for detecting EML4-ALK fusion comprise combinations of V1, V2, V3a, V3b, V5a, V5b, V5'and V6. And the detection sites for detecting the ROS1 fusion comprise a combination of SDC4-ROS1S2: R32, SLC34A2-ROS1SL4: R32, CD74-ROS1C6: R32, CD74-ROS1C6: R34, EZR ROSE10: R34, GOPC-ROS1G8: R35 and TPM3-ROS1T8: R35, and the detection sites for detecting the ROS1 fusion comprise a combination of SDC4-ROS1S2: R32, SLC34A2-ROS1SL4: R32, CD74-ROS1C6: R32, CD74-ROS1C6: R34, according to the invention, RNA in a plasma exosome is separated, and multiple joint detection of EML4-ALK and ROS1 fusion is simultaneously completed in a single tube by virtue of a qRT-PCR platform.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a primer-probe combination for jointly detecting EML4-ALK fusion and ROS1 fusion in plasma exosomes and an application thereof. Background Art

[0002] In non-small cell lung cancer (NSCLC), the activation of the anaplastic lymphoma kinase (ALK) gene and another lung cancer driver gene ROS1 is mainly caused by gene rearrangement. ROS1 and ALK have nearly 49% similarity in amino acid sequence, and the homology of ATP binding site in the kinase catalytic region is as high as 77%. The clinical features of lung cancers of the two genotypes of ALK and ROS1 are also very similar.

[0003] The incidence of ALK fusion in non-small cell lung cancer is about 5.6%, of which the incidence in adenocarcinoma is 6.6%-9.6%. ALK mutation in lung cancer is mainly the breakage and rearrangement of ALK gene with other genes. Among them, echinoderm microtubule-binding protein 4-ALK (EML4-ALK) fusion gene mutation is the main type, accounting for 85% of ALK fusion; the positive rate of ROS1 fusion gene in non-small cell lung cancer is 1.0%-3.4%.

[0004] When ALK and EML4 fuse, all ALK gene fusions occur in a sequence encoded by exon 20, while the EML4 breakpoints show diversity. The breakpoints that have been detected include exons 2 / 6 / 13 / 14 / 15 / 17 / 18 / 20, for a total of 8 common fusion types.

[0005] The ROS1 gene is located at 6q22.1, and multiple partner genes can rearrange with ROS1 to activate the gene. ROS1 fusion often occurs in exons 32 / 34 / 35 / 36. Among them, the most common fusion gene subtype is CD74-ROS1, accounting for about 30%, followed by EZR-ROS1. There are 7 common fusion types in NSCLC.

[0006] Currently, three generations of ALK inhibitors have been approved for marketing, including crizotinib, alectinib, ceritinib, brigatinib, and lorlatinib. Different ALK inhibitors show different activities against the ROS1 gene, among which crizotinib is the earliest approved small molecule inhibitor targeting the ROS1 target. Recent clinical studies have shown that ceritinib and lorlatinib also have good clinical responses to ROS1 fusion-positive, while alectinib does not show inhibitory activity. In addition, entrectinib has been approved for the treatment of ROS1 fusion-positive, and several new drugs targeting ROS1 fusion, such as lopatinib, are under development.

[0007] Among the existing ALK and ROS1 detection methods, whether IHC, FISH, qPCR or NGS, they are mainly based on tissue samples for detection. In clinical detection, 75% of lung cancer patients are already in the middle and late stages at the time of initial diagnosis and cannot be cured by surgery. It is challenging to obtain tissue biopsies from NSCLC, and up to 30% of patients do not have tissues for genetic molecular analysis. The cell-free nucleic acids (exosomes, free circulating cfDNA) shed by tumor cells carry rich tumor molecular information. Liquid biopsy can be used as an important supplementary means of detecting patients with advanced NSCLC.

[0008] Exosomes, also known as exosomes, are 30-150nm in diameter. They are small vesicles wrapped in lipid bilayer membranes that are released into the extracellular environment after the fusion of intracellular multivesicular bodies (MVBs) with the cell membrane. Exosomes contain a variety of nucleic acids (DNA, mRNA, microRNA (miRNA), lncRNA, circRNA, etc.) and proteins.

[0009] Blood tests are currently mainly based on EGFR mutation detection in cfDNA samples, and fusion tests in liquid biopsies are mainly single ALK fusion or ROS1 fusion tests. In the relevant diagnosis and treatment guidelines for non-small cell lung cancer, ALK and ROS1 tests are also recommended for small biopsy specimens or non-smoking squamous cell carcinoma patients (Class I recommendation). Therefore, a convenient and accurate ALK / ROS1 detection method is needed to meet clinical needs.

[0010] Currently, the main ALK tests include FISH, Ventana IHC, RT-PCR and digital PCR. The main ROS1 tests are FISH and RT-PCR. Both IHC and FISH tests require tissue samples, and the currently available RT-PCR test kits also mainly use tissue samples. For patients with advanced or metastatic NSCLC who need ALK and ROS1 testing and analysis, small samples or liquid biopsies are more feasible than tissue samples. In addition, current diagnostic and treatment guidelines recommend that patients with lung adenocarcinoma should undergo combined testing of genes such as ALK and ROS1 to avoid waste of samples and the inconvenience caused to patients by repeated sampling.

[0011] There are attempts to use digital PCR to detect ALK and ROS1 fusions, and even use cfRNA as a detection template. However, the digital PCR detection steps are cumbersome and costly, and it is not as fast as qPCR detection. Compared with exosomes, exRNA is wrapped in protein, and the stability of cfRNA against RNase is far less than that of exosomes. In addition, cfRNA storage tubes are required when blood is collected, but the blood collection tubes used are expensive. In addition, the components used to stabilize RNA are prone to denaturation of proteins and other substances in the blood and cross-linking of nucleic acids, which are not easy to remove during the extraction process, thus affecting downstream applications. Therefore, there is an urgent need to develop a set of methods suitable for the combined detection of fusion genes in exosome RNA. Summary of the invention

[0012] In view of the shortcomings of the prior art, the object of the present invention is to provide a primer probe combination and application thereof for the joint detection of EML4-ALK fusion and ROS1 fusion in plasma exosomes. The present invention provides a set of primer probes and detection reagents for detecting EML4-ALK fusion and ROS1 fusion in human plasma exosomes. The present invention separates RNA from plasma exosomes and uses a qRT-PCR platform to simultaneously complete multiple joint detection of EML4-ALK and ROS1 fusion in a single tube.

[0013] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0014] In a first aspect, the present invention provides a primer-probe combination for jointly detecting EML4-ALK fusion and ROS1 fusion in plasma exosomes, wherein the primer-probe combination comprises a primer pair and a probe for detecting EML4-ALK fusion, and a primer pair and a probe for detecting ROS1 fusion;

[0015] Assay sites for detecting EML4-ALK fusions include a combination of EML4-ALK V1, EML4-ALK V2, EML4-ALK V3a, EML4-ALK V3b, EML4-ALK V5a, EML4-ALK V5b, EML4-ALK V5′, and EML4-ALK V6;

[0016] The assay sites for detecting ROS1 fusions included a combination of SDC4-ROS1_S2:R32, SLC34A2-ROS1_SL4:R32, CD74-ROS1_C6:R32, CD74-ROS1_C6:R34, EZR ROS_E10:R34, GOPC-ROS1_G8:R35, and TPM3-ROS1_T8:R35.

[0017] In the present invention, the primer-probe combination can detect 8 common ALK fusions and 7 ROS1 fusions at one time. For patients in the late stage or for whom tissue samples are not suitable, detection can be achieved by collecting peripheral blood from patients; a single tube test can cover 8 common ALK fusions and 7 ROS1 fusions, with low sample requirements and high detection throughput.

[0018] In the present invention, the same primer pair is used to detect two ALK fusions, EML4-ALK V1 and EML4-ALK V6. Both EML4-ALK V1 and EML4-ALK V6 are formed by the fusion of EML4 exon 13 and ALK exon 20. EML4-ALK V6 has a 69 bp insertion fragment at the breakpoint.

[0019] Preferably, the forward primer for detecting EML4-ALK V1 and EML4-ALK V6 is shown as SEQ ID NO:1, and the reverse primer is shown as SEQ ID NO:6.

[0020] Preferably, the forward primer for detecting EML4-ALK V2 is shown as SEQ ID NO:2, and the reverse primer is shown as SEQ ID NO:6.

[0021] Preferably, the forward primer for detecting EML4-ALK V3a and EML4-ALK V3b is shown as SEQ ID NO:3, and the reverse primer is shown as SEQ ID NO:6.

[0022] In the present invention, the same primer pair is used to detect two ALK fusions, EML4-ALK V3a and EML4-ALK V3b. Both EML4-ALK V3a and EML4-ALK V3b are formed by the fusion of EML4 exon 6 and ALK exon 20. EML4-ALK V3b has a 33 bp insertion fragment at the breakpoint.

[0023] Preferably, the forward primer for detecting EML4-ALK V5a and EML4-ALK V5b is shown as SEQ ID NO:4, and the reverse primer is shown as SEQ ID NO:6.

[0024] In the present invention, the same primer pair is used to detect the two ALK fusions EML4-ALK V5a and EML4-ALK V5b. Both EML4-ALK V5a and EML4-ALK V5b are formed by the fusion of EML4 exon 2 and ALK exon 20. EML4-ALK V5b has a 117 bp insertion fragment at the breakpoint.

[0025] Preferably, the forward primer for detecting EML4-ALK V5' is shown as SEQ ID NO:5, and the reverse primer is shown as SEQ ID NO:6.

[0026] Preferably, the probe for detecting EML4-ALK fusion is as shown in SEQ ID NO:7.

[0027] In the present invention, when detecting the detection sites of EML4-ALK fusion, the reverse primers used for the above 8 detection sites are reverse universal primers, which simplifies the primer design and reduces the number of primers.

[0028] Preferably, the forward primer for detecting SDC4-ROS1_S2:R32 is shown as SEQ ID NO:8, and the reverse primer is shown as SEQ ID NO:11.

[0029] Preferably, the forward primer for detecting SLC34A2-ROS1_SL4:R32 is shown as SEQ ID NO:9, and the reverse primer is shown as SEQ ID NO:11.

[0030] Preferably, the forward primer for detecting CD74-ROS1_C6:R32 is shown as SEQ ID NO:10, and the reverse primer is shown as SEQ ID NO:11.

[0031] Preferably, the probe for detecting SDC4-ROS1_S2:R32, SLC34A2-ROS1_SL4:R32 and CD74-ROS1_C6:R32 is as shown in SEQ ID NO:12.

[0032] In the present invention, the reverse primers for detecting sites such as SDC4-ROS1_S2:R32, SLC34A2-ROS1_SL4:R32 and CD74-ROS1_C6:R32 are the same reverse universal primer.

[0033] Preferably, the forward primer for detecting CD74-ROS1_C6:R34 is shown as SEQ ID NO:13, and the reverse primer is shown as SEQ ID NO:15.

[0034] Preferably, the forward primer for detecting EZR ROS_E10:R34 is shown as SEQ ID NO:14, and the reverse primer is shown as SEQ ID NO:15.

[0035] Preferably, the probe for detecting CD74-ROS1_C6:R34 and EZR ROS_E10:R34 is as shown in SEQ ID NO:16.

[0036] In the present invention, the reverse primers for detecting sites such as CD74-ROS1_C6:R34 and EZR ROS_E10:R34 are the same reverse universal primer.

[0037] Preferably, the forward primer for detecting GOPC-ROS1_G8:R35 is shown as SEQ ID NO:17, and the reverse primer is shown as SEQ ID NO:19.

[0038] Preferably, the forward primer for detecting TPM3-ROS1_T8:R35 is shown as SEQ ID NO:18, and the reverse primer is shown as SEQ ID NO:19.

[0039] Preferably, the probe for detecting GOPC-ROS1_G8:R35 and TPM3-ROS1_T8:R35 is as shown in SEQ ID NO:20.

[0040] In the present invention, the reverse primers for detecting sites such as GOPC-ROS1_G8:R35 and TPM3-ROS1_T8:R35 are the same reverse universal primer.

[0041] Preferably, the primer-probe combination further comprises a primer pair and a probe for detecting an internal reference gene HPRT.

[0042] Preferably, the forward primer for detecting HPRT is shown as SEQ ID NO:21, and the reverse primer is shown as SEQ ID NO:23.

[0043] Preferably, the probe for detecting HPRT is as shown in SEQ ID NO:22.

[0044] The internal reference gene used in the present invention is a housekeeping gene, which can be stably detected in plasma and can well monitor the false negative of the entire reaction system.

[0045] In the present invention, the high incompleteness of the nucleic acid in the exosome sample is taken into consideration when designing the primers, ensuring that the length of the amplicon is as short as possible, the positions of the primers and the probe are concentrated as close to the breakpoint as possible, and the Tm value is slightly lower than the primer design of the tissue or blood sample by 2-5°C. Under the premise of ensuring the detection specificity, it can also ensure that more template sequences can effectively bind to the primers and probes.

[0046] Preferably, the 5' end of the probe carries a fluorescent group, and the 3' end of the probe carries a quenching group.

[0047] Preferably, the fluorescent group of the probe for detecting EML4-ALK fusion and ROS1 fusion is selected from any one or two of FAM, VIC, TAMRA, ROX, CY5 or CY5.5.

[0048] Preferably, the quenching group of the probe for detecting EML4-ALK fusion and ROS1 fusion is selected from any one or two of MGB, BHQ1, BHQ2, BHQ3, SQ1, SQ2 or SQ3.

[0049] Preferably, the fluorescent group of the probe for detecting the internal reference gene HPRT is selected from any one of FAM, VIC, TAMRA, ROX, CY5 or CY5.5.

[0050] Preferably, the quenching group of the probe for detecting the internal reference gene HPRT is selected from any one of MGB, BHQ1, BHQ2, BHQ3, SQ1, SQ2 or SQ3.

[0051] In the present invention, the fluorescent groups and quenching groups of the probes for detecting EML4-ALK fusion and ROS1 fusion can be the same or different. When the fluorescent groups of different probes are different, different fusion types can also be distinguished.

[0052] In a second aspect, the present invention provides a kit for jointly detecting EML4-ALK fusion and ROS1 fusion in plasma exosomes, wherein the kit comprises the primer-probe combination for jointly detecting EML4-ALK fusion and ROS1 fusion in plasma exosomes described in the first aspect.

[0053] Preferably, the kit further comprises a detection reagent, and the detection reagent comprises a reaction solution A and a reaction solution B.

[0054] Preferably, the reaction solution A comprises, by molar concentration, 0.1-50 mM dATP (for example, 0.1 mM, 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM or 50 mM, etc.), 0.1-50 mM dCTP (for example, 0.1 mM, 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM or 50 mM, etc.), 0.1-50 mM dGTP (for example, 0.1 mM, 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM or 50 mM, etc.), 0.1-100 mM dUTP (for example, 0.1 mM, 10 mM, 20 mM, 40 mM, 60 mM, 80 mM or 100 mM, etc.), 1-500 mM MgSO 4 (for example, it can be 1mM, 50mM, 100mM, 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM or 500mM, etc.) and 1-500mM Tris-HCl (for example, it can be 1mM, 50mM, 100mM, 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM or 500mM, etc.).

[0055] Preferably, the reaction solution B comprises the following components: reverse transcriptase, RNase inhibitor, hot-start DNA polymerase and thermosensitive UDG enzyme.

[0056] In a third aspect, the present invention provides a device for jointly detecting EML4-ALK fusion and ROS1 fusion in plasma exosomes, the device comprising:

[0057] PCR amplification module: using the primer-probe combination in the kit for joint detection of EML4-ALK fusion and ROS1 fusion in plasma exosomes described in the second aspect to prepare a PCR system, and performing PCR amplification on the sample to be tested;

[0058] Result analysis module: The Ct value is obtained by fluorescence quantitative PCR detection, and EML4-ALK fusion and ROS1 fusion are judged according to the Ct value.

[0059] Preferably, the final concentrations of primers for detecting EML4-ALK fusion and ROS1 fusion in the PCR system are each independently 100-700 nM, for example, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM or 700 nM, etc.; the final concentrations of probes are each independently 25-400 nM, for example, 25 nM, 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM or 400 nM, etc.

[0060] Preferably, the final concentration of the primers for detecting the internal reference gene HPRT in the PCR system is independently 100-700 nM, for example, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM or 700 nM, etc.; the final concentration of the probe is 25-400 nM, for example, 25 nM, 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM or 400 nM, etc.

[0061] Preferably, the components in the reaction solution B in the PCR system include, by final concentration: 1-1000U / 50μL reverse transcriptase (for example, 1U / 50μL, 5U / 50μL, 100U / 50μL, 300U / 50μL, 500U / 50μL, 700U / 50μL, 900U / 50μL or 1000U / 50μL, etc.), 1-500U / 50μL RNase inhibitor (e.g., 1U / 50μL, 50U / 50μL, 100U / 50μL, 200U / 50μL, 300U / 50μL, 400U / 50μL, or 500U / 50μL), 1-500U / 50μL hot start DNA polymerase (e.g., 1U / 50μL, 50U / 50μL, 100U / 50μL, 200U / 50μL, 300U / 50μL, 400U / 50μL, or 500U / 50μL), U / 50μL, 300U / 50μL, 400U / 50μL or 500U / 50μL, etc.) and 0.1-100U / 50μL thermosensitive UDG enzyme (for example, it can be 0.1U / 50μL, 1U / 50μL, 10U / 50μL, 30U / 50μL, 50U / 50μL, 70U / 50μL, 90U / 50μL or 100U / 50μL, etc.).

[0062] In a fourth aspect, the present invention provides the use of the primer-probe combination for the joint detection of EML4-ALK fusion and ROS1 fusion in plasma exosomes described in the first aspect and / or the kit for the joint detection of EML4-ALK fusion and ROS1 fusion in plasma exosomes described in the second aspect in the preparation of a product for detecting lung cancer.

[0063] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0064] Compared with the prior art, the present invention has the following beneficial effects:

[0065] The present invention can detect 8 common ALK fusions and 7 ROS1 fusions at one time. The internal reference gene used in the present invention is a housekeeping gene, which can also be stably detected in plasma, and can well monitor the false negative of the entire reaction system. When designing primers, the high incompleteness of the nucleic acid in the exosome sample is taken into account to ensure that the amplicon length is as short as possible, and the positions of the primers and probes are concentrated near the breakpoint as much as possible. At the same time, the Tm value is slightly lower than the primer design of tissue or blood samples by 2-5°C. Under the premise of ensuring the specificity of the detection, it can also ensure that more template sequences can effectively bind to the primers and probes. The primer probe and kit have the following advantages: first, for patients in the advanced stage or for whom tissue samples are not suitable, detection can be achieved by collecting peripheral blood from patients; second, a single tube test can cover 8 common ALK fusions and 7 ROS1 fusions, with low sample requirements and high detection throughput; third, a single test can distinguish between ALK fusion and ROS1 fusion, providing a basis for subsequent precise drug delivery; fourth, the internal reference gene used is a housekeeping gene, which can also be stably detected in plasma, and can well monitor the false negatives of the entire reaction system. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 These are the PCR detection results of EML4-ALK V1 forward primer F1 and EML4-ALK V1 forward primer F2.

[0067] Figure 2 These are the PCR detection results of EML4-ALK V3a&V3b forward primer F1 and EML4-ALK V3a&V3b forward primer F2.

[0068] Figure 3 These are the PCR detection results of SDC4-ROS1 forward primer F1 and SDC4-ROS1 forward primer F2.

[0069] Figure 4 These are the PCR detection results of CD74-ROS1_C6:R34 forward primer F1 and CD74-ROS1_C6:R34 forward primer F2.

[0070] Figure 5 The results of PCR detection of EML4-ALK V1 in exosomes from the supernatant of H3122 and H293T cells.

[0071] Figure 6 The results of PCR detection of HPRT in exosomes from the supernatant of H3122 and H293T cells.

[0072] Figure 7 This is the result of EML4-ALK V3 fusion detection in exosomes from H2228 cell supernatant.

[0073] Figure 8 This is the detection result of CD74-ROS1 fusion in the exosomes of CD74-ROS1 / BaF3 cell supernatant.

[0074] Fig. 9 This is the detection result of HPRT in the exosomes of CD74-ROS1 / BaF3 cell supernatant.

[0075] Fig.10 Amplification results for ALK-positive patients.

[0076] Fig.11 Amplification results for ROS1-positive patients.

[0077] Fig.12 Shown are the HPRT amplification results for all samples. DETAILED DESCRIPTION

[0078] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only used to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0079] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0080] Example 1

[0081] This embodiment provides a primer probe for detecting the combined detection of EML4-ALK fusion and ROS1 fusion and a detection method thereof.

[0082] Experimental materials: EML4-ALK V1 fusion plasmid, EML4-ALK V3a fusion plasmid, EML4-ALK V3b fusion plasmid, SDC4-ROS1_S2:R32 fusion plasmid, CD74-ROS1_C6:R34 fusion plasmid. The EML4-ALK V1 fusion plasmid (EAV1) integrates a DNA sequence of EML4 exon 13 and ALK exon 20. The EML4-ALK V3a fusion plasmid (EAV3a) integrates a DNA sequence of EML4 exon 6 and ALK exon 20. The SDC4-ROS1_S2:R32 fusion plasmid (S2R32) integrates a DNA sequence of SDC4 gene exon 2 and ROS1 gene exon 32. The CD74-ROS1_C6:R34 fusion plasmid (C6R34) integrates a DNA sequence of the 6th exon of the CD74 gene and the 34th exon of the ROS1 gene.

[0083] The experimental steps are as follows:

[0084] 1.1 Prepare the PCR amplification system and transfer to ABI 7500 PCR instrument for amplification.

[0085] 1.1.1 EML4-ALK V1 (hereinafter referred to as "V1", the remaining detection sites are referred to as V2, V3a, V3b, V5a, V5b, V5' and V6) in Table 1, V6 forward primer F1 (SEQ ID NO: 1), 1, V6 forward primer F2 (SEQ ID NO: 24); V3a, V3b forward primer F1 (SEQ ID NO: 3), V3a, V3b forward primer F2 (SEQ ID NO: 25); SDC4-ROS1_S2:R32 forward primer F1 (SEQ ID NO: 8), SDC4-ROS1_S2:R32 forward primer F2 (SEQ ID NO: 26), CD74-ROS1_C6:R34 forward primer F1 (SEQ ID NO: 13), CD74-ROS1_C6:R34 forward primer F2 (SEQ ID NO: ID NO: 27) powder was diluted according to the synthesis instructions and mixed into a primer-probe mixture.

[0086] Table 1

[0087]

[0088]

[0089] The EML4-ALK V1&V6 forward primers in Table 1 refer to forward primers used for simultaneous detection of EML4-ALK V1 and EML4-ALK V6.

[0090] The probes in Table 1 also have fluorescent groups and quenching groups, as shown below: The fluorescent group of the EML4-ALK probe is FAM, and the quenching group is BHQ1. The fluorescent group of the ROS1 E32 universal probe is ROX, and the quenching group is BHQ2. The fluorescent group of the ROS1E34 universal probe is ROX, and the quenching group is BHQ2. The fluorescent group of the ROS1 E35 universal probe is ROX, and the quenching group is BHQ2. The fluorescent group of the HPRT probe is VIC, and the quenching group is BHQ1.

[0091] 1.1.2 The reaction system was prepared as follows: 18 μL of PCR reaction solution A, containing dATP, dGTP, and dCTP at a final concentration of 0.2 mM, dUTP at a final concentration of 0.4 mM, KCl 50 mM, Tris-HCl 10 mM, MgSO 42.5mM; 3μL of enzyme mixture B, containing 0.1U UDG, 5U RNase inhibitor, 5U Taq enzyme antibody, 5U Taq DNA polymerase, and 20U reverse transcriptase; 1μL of primer-probe mixture for detecting EML4-ALK fusion, detecting ROS1 fusion, and detecting HPRT, the concentration of F primers in the reaction system was 300nM, the concentration of R primers in the mixture was 500nM, and the concentration of probes was 200nM.

[0092] 1.1.3 Add 20 μL of plasmid DNA to make the final volume 50 μL.

[0093] 1.1.4 The amplification program was as follows: reverse transcription at 50°C for 10 minutes, pre-denaturation at 95°C for 2 minutes, 45 cycles (95°C for 5s; 60°C for 35s), and fluorescence was collected at 60°C.

[0094] PCR results were detected as Figure 1-Figure 4 As shown: Figure 1 These are the PCR detection results of EML4-ALK V1&V6 forward primer F1 and EML4-ALK V1&V6 forward primer F2. Figure 2 These are the PCR detection results of EML4-ALK V3a&V3b forward primer F1 and EML4-ALK V3a&V3b forward primer F2. Figure 3 These are the PCR detection results of SDC4-ROS1_S2:R32 forward primer F1 and SDC4-ROS1_S2:R32 forward primer F2. Figure 4 These are the PCR detection results of CD74-ROS1_C6:R34 forward primer F1 and CD74-ROS1_C6:R34 forward primer F2.

[0095] from Figures 1 to 4 It can be seen that when detecting EAV1 plasmid, the amplification efficiency of EML4-ALK V1&V6 forward primer F1 is better than that of EML4-ALK V1&V6 forward primer F2; when detecting EAV3a plasmid, the amplification efficiency of EML4-ALK V3a&V3b forward primer F1 is better than that of EML4-ALK V3a&V3b forward primer F2; when detecting SDC4-ROS1_S2:R32 fusion plasmid S2R32, the amplification efficiency of SDC4-ROS1_S2:R32 forward primer F1 is better than that of SDC4-ROS1_S2:R32 forward primer F2; when detecting CD74-ROS1_C6:R34 fusion plasmid C6R34, the amplification efficiency of CD74-ROS1_C6:R34 forward primer F1 is better than that of CD74-ROS1_C6:R34 forward primer F2.

[0096] Example 2

[0097] In this example, the primers and probes in Example 1 were used to detect exosomes in the supernatant of H3122 cell line and H293T cell line (negative control).

[0098] Experimental materials: Exosomes from the supernatant of H3122 (EML4-ALK V1 positive) cell line and H293T cell line (negative control).

[0099] The experimental steps are as follows:

[0100] 2.1 Collect 20 mL of supernatant from H3122 and H293T cells after 48 hours of serum-free culture.

[0101] 2.2 Centrifuge at 4700g and 4°C for 30 minutes to remove cell debris.

[0102] 2.3 Add 10 mL of total exosome isolation reagent (Invitrogen, 4478359) and precipitate at 4°C overnight.

[0103] 2.4 Centrifuge at 4700g and 4°C for 15 minutes, discard the supernatant, add 300μL PBS, and mix by pipetting.

[0104] 2.5 RNA was extracted using an RNA extraction kit (RNeasy Micro Kit, 74004) and eluted with 60 μL of elution buffer.

[0105] 2.6 Prepare the PCR amplification system and transfer to ABI 7500 PCR instrument for amplification.

[0106] 2.6.1 The primer and probe powders for detecting EML4-ALK fusion and HPRT in Table 1 were diluted according to the synthesis instructions, and mixed into primer and probe mixtures.

[0107] 2.6.2 The reaction system was prepared as follows: 18 μL of PCR reaction solution A, containing dATP, dGTP, and dCTP at a final concentration of 0.2 mM, dUTP at a final concentration of 0.4 mM, KCl 50 mM, Tris-HCl 10 mM, MgSO 4 2.5mM; 3μL of enzyme mixture B, containing 0.1U UDG, 5U RNase inhibitor, 5U Taq enzyme antibody, 5U Taq DNA polymerase, and 20U reverse transcriptase; 1μL of primer-probe mixture for detecting EML4-ALK fusion, detecting ROS1 fusion, and detecting HPRT, the concentration of F primers in the reaction system was 300nM, the concentration of R primers in the mixture was 500nM, and the concentration of probes was 200nM.

[0108] 2.6.3 Add 20 μL RNA to make the final volume 50 μL.

[0109] 2.6.4 The amplification program was as follows: reverse transcription at 50°C for 10 minutes, pre-denaturation at 95°C for 2 minutes; 45 cycles (95°C, 5s; 60°C, 35s), and fluorescence was collected at 60°C.

[0110] PCR results were detected as Figure 5 and Figure 6 As shown: Figure 5 The results of PCR detection of EML4-ALKV1 in exosomes from the supernatant of H3122 and H293T cells. Figure 6 These are the PCR detection results of the internal reference gene HPRT in the exosomes of H3122 and H293T cell supernatants.

[0111] In the exosomes of H3122, the FAM channel is EML4-ALK V1 fusion, while in the control H293T, only the internal reference gene of the VIC channel can be detected. The above results show that the primer probe combination can accurately detect EML4-ALK fusion in the exosomes of the supernatant of positive cell lines.

[0112] Example 3

[0113] In this example, the primers and probes in Example 1 were used to detect exosomes in the supernatant of H2228 (EML4-ALK V3 positive) cell line.

[0114] Experimental materials: Exosomes from the supernatant of H2228 cell line.

[0115] The experimental steps are as follows:

[0116] 3.1 Collect 20 mL of H2228 cell supernatant after 48 hours of serum-free culture.

[0117] 3.2 Centrifuge at 4700g and 4°C for 30 minutes to remove cell debris.

[0118] 3.3 Add 10 mL of total exosome isolation reagent (Invitrogen, 4478359) and precipitate at 4°C overnight.

[0119] 3.4 Centrifuge at 4700g and 4℃ for 15 minutes, discard the supernatant, add 300μL PBS, and mix by pipetting.

[0120] 3.5 RNA was extracted using an RNA extraction kit (RNeasy Micro Kit, 74004) and eluted with 60 μL of elution buffer.

[0121] 3.6 Prepare the PCR amplification system and transfer to ABI 7500 PCR instrument for amplification.

[0122] 3.6.1 The primer and probe powders for detecting EML4-ALK fusion and HPRT in Table 1 were diluted according to the synthesis instructions, and mixed into primer and probe mixtures.

[0123] 3.6.2 The reaction system was prepared as follows: 18 μL of PCR reaction solution A, containing dATP, dGTP, and dCTP at a final concentration of 0.2 mM, dUTP at a final concentration of 0.4 mM, KCl 50 mM, Tris-HCl 10 mM, MgSO 4 2.5mM; 3μL of enzyme mixture B, containing 0.1U UDG, 5U RNase inhibitor, 5U Taq enzyme antibody, 5U Taq DNA polymerase, and 20U reverse transcriptase; 1μL of primer-probe mixture for detecting EML4-ALK fusion, detecting ROS1 fusion, and detecting HPRT, the concentration of F primers in the reaction system was 300nM, the concentration of R primers in the mixture was 500nM, and the concentration of probes was 200nM.

[0124] 3.6.3 Add 20 μL RNA to make the final volume 50 μL.

[0125] 3.6.4 The amplification program was as follows: reverse transcription at 50°C for 10 minutes, pre-denaturation at 95°C for 2 minutes; 45 cycles (95°C, 5s; 60°C, 35s), and fluorescence was collected at 60°C.

[0126] PCR results were detected as Figure 7 As shown: Figure 7 The results of EML4-ALK V3 fusion detection in H2228 cell supernatant exosomes show that EML4-ALK V3 fusion can be detected in H2228 exosomes.

[0127] Example 4

[0128] In this example, the primer probe in Example 1 was used to detect CD74-ROS1 fusion in the exosomes of CD74-ROS1 / BaF3 cell supernatant.

[0129] Experimental materials: Exosomes from the supernatant of CD74-ROS1 / BaF3 cell line.

[0130] The experimental steps are as follows:

[0131] 4.1 Collect 20 mL of supernatant from CD74-ROS1 / BaF3 cells after 48 hours of serum-free culture.

[0132] 4.2 Centrifuge at 4700g and 4°C for 30 minutes to remove cell debris.

[0133] 4.3 Add 10 mL of total exosome isolation reagent (Invitrogen, 4478359) and precipitate at 4°C overnight.

[0134] 4.4 Centrifuge at 4700g and 4℃ for 15 minutes, discard the supernatant, add 300μL PBS, and mix by pipetting.

[0135] 4.5 RNA was extracted using an RNA extraction kit (RNeasy Micro Kit, 74004) and eluted using 60 μL of elution buffer.

[0136] 4.6 Prepare the PCR amplification system and transfer to ABI 7500 PCR instrument for amplification.

[0137] 4.6.1 Dilute the primer and probe powders for detecting ROS1 fusion and HPRT in Table 1 according to the synthesis instructions, and mix them into primer and probe mixtures.

[0138] 4.6.2 The reaction system was prepared as follows: 18 μL of PCR reaction solution A, containing dATP, dGTP, and dCTP at a final concentration of 0.2 mM, dUTP at a final concentration of 0.4 mM, KCl 50 mM, Tris-HCl 10 mM, MgSO 4 2.5mM; 3μL of enzyme mixture B, containing 0.1U UDG, 5U RNase inhibitor, 5U Taq enzyme antibody, 5U Taq DNA polymerase, and 20U reverse transcriptase; 1μL of primer-probe mixture for detecting EML4-ALK fusion, detecting ROS1 fusion, and detecting HPRT, the concentration of F primers in the reaction system was 300nM, the concentration of R primers in the mixture was 500nM, and the concentration of probes was 200nM.

[0139] 4.6.3 Add 20 μL RNA to make the final volume 50 μL.

[0140] 4.6.4 The amplification program was as follows: reverse transcription at 50°C for 10 minutes, pre-denaturation at 95°C for 2 minutes; 45 cycles (95°C, 5s; 60°C, 35s), and fluorescence was collected at 60°C.

[0141] PCR results were detected as Figure 8 and Fig. 9 As shown: Figure 8 This is the detection result of CD74-ROS1 fusion in the exosomes of CD74-ROS1 / BaF3 cell supernatant. Fig. 9The results of HPRT detection in CD74-ROS1 / BaF3 cell supernatant exosomes show that CD74-ROS1 fusion can be successfully detected in the ROX channel in CD74-ROS1 / BaF3 cell supernatant exosomes, and HPRT is an internal reference in the VIC channel.

[0142] Example 5

[0143] This example uses the primers and probes in Example 1 to jointly detect ALK / ROS1 fusion in plasma exosomes of healthy subjects and EML4-ALK positive and ROS1 fusion positive subjects.

[0144] Experimental materials: EDTA plasma from 4 healthy subjects, plasma from 2 EML4-ALK V1 positive (Case 1, Case 2), and plasma from 1 CD74-ROS1 positive (Case 3) lung cancer patients.

[0145] The experimental steps are as follows:

[0146] 5.1 Take the plasma sample and centrifuge it at 16000g and 4℃ for 10 minutes. Take 2 mL of the supernatant and set aside.

[0147] 5.2 Add 0.5 mL of exosome rapid precipitation solution (SBI, EXOQ20A-1) and vortex to mix.

[0148] 5.3 Centrifuge the mixed plasma at 3000g and 4°C for 10 min, discard the supernatant, and add 1 mL of PBS to resuspend the exosome pellet.

[0149] 5.4 Add 0.25 mL of ExoQuick exosome rapid precipitation solution (SBI, EXOQ20A-1) and vortex to mix.

[0150] 5.5 The mixed exosome resuspension was centrifuged at 3000g and 4°C for 10 minutes. The supernatant was discarded and 0.1 mL PBS was added to resuspend the exosome pellet.

[0151] 5.6 Extract RNA using RNA extraction kit (RNeasy Micro Kit, 74004) and elute with 60 μL elution buffer.

[0152] 5.7 Prepare PCR amplification system and transfer to ABI 7500 PCR instrument for amplification.

[0153] 5.7.1 The primer and probe powders for detecting EML4-ALK fusion, detecting ROS1 fusion, and detecting HPRT in Table 1 were diluted according to the synthesis instructions, and mixed into primer and probe mixtures.

[0154] 5.7.2 The reaction system was prepared as follows: 18 μL of PCR reaction solution A, containing dATP, dGTP, and dCTP at a final concentration of 0.2 mM, dUTP at a final concentration of 0.4 mM, KCl 50 mM, Tris-HCl 10 mM, MgSO 4 2.5mM; 3μL of enzyme mixture B, containing 0.1U UDG, 5U RNase inhibitor, 5U Taq enzyme antibody, 5U Taq DNA polymerase, and 20U reverse transcriptase; 1μL of primer-probe mixture for detecting EML4-ALK fusion, detecting ROS1 fusion, and detecting HPRT, the concentration of F primers in the reaction system was 300nM, the concentration of R primers in the mixture was 500nM, and the concentration of probes was 200nM.

[0155] 5.7.3 Add 20 μL RNA to make the final volume 50 μL.

[0156] 5.7.4 The amplification program was as follows: reverse transcription at 50°C for 10 minutes, pre-denaturation at 95°C for 2 minutes; 45 cycles (95°C, 5s; 60°C, 35s), and fluorescence was collected at 60°C.

[0157] PCR results were detected as Figure 10-12 As shown: Fig.10 Amplification results for ALK-positive patients. Fig.11 Amplification results for ROS1-positive patients. Fig.12 The HPRT amplification results of all samples. The amplification results show that positive signals can be detected in the plasma exosomes of 2 ALK fusion-positive patients and 1 ROS1 fusion-positive patient. The internal reference HPRT can be detected in all samples.

[0158] In summary, the primer probe for detecting EML4-ALK fusion and ROS1 fusion described in the present invention can realize the one-time detection of 8 common ALK fusions and 7 ROS1 fusions. A single-tube test can cover common fusions, with low sample demand, high detection throughput, and accurate test results. It has important application value in the detection of non-small cell lung cancer.

[0159] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A primer-probe combination for the joint detection of EML4-ALK fusion and ROS1 fusion in plasma exosomes, It is characterized in that The primer-probe combination includes a primer pair and a probe for detecting EML4-ALK fusion, and a primer pair and a probe for detecting ROS1 fusion; Assay sites for detecting EML4-ALK fusions include a combination of EML4-ALK V1, EML4-ALK V2, EML4-ALK V3a, EML4-ALK V3b, EML4-ALK V5a, EML4-ALK V5b, EML4-ALK V5′, and EML4-ALK V6; The assay sites for detecting ROS1 fusions included a combination of SDC4-ROS1_S2:R32, SLC34A2-ROS1_SL4:R32, CD74-ROS1_C6:R32, CD74-ROS1_C6:R34, EZR ROS_E10:R34, GOPC-ROS1_G8:R35, and TPM3-ROS1_T8:R35.

2. The primer-probe combination for the joint detection of EML4-ALK fusion and ROS1 fusion in plasma exosomes according to claim 1, It is characterized in that The forward primer for detecting EML4-ALK V1 and EML4-ALK V6 is shown in SEQ ID NO: 1, and the reverse primer is shown in SEQ ID NO: 6; Preferably, the forward primer for detecting EML4-ALK V2 is shown as SEQ ID NO: 2, and the reverse primer is shown as SEQ ID NO: 6; Preferably, the forward primer for detecting EML4-ALK V3a and EML4-ALK V3b is shown in SEQ ID NO:3, and the reverse primer is shown in SEQ ID NO:6; Preferably, the forward primer for detecting EML4-ALK V5a and EML4-ALK V5b is shown in SEQ ID NO:4, and the reverse primer is shown in SEQ ID NO:6; Preferably, the forward primer for detecting EML4-ALK V5' is shown as SEQ ID NO:5, and the reverse primer is shown as SEQ ID NO:6; Preferably, the probe for detecting EML4-ALK fusion is as shown in SEQ ID NO:

7.

3. The primer-probe combination for the joint detection of EML4-ALK fusion and ROS1 fusion in plasma exosomes according to claim 1 or 2, It is characterized in that The forward primer for detecting SDC4-ROS1_S2:R32 is shown in SEQ ID NO:8, and the reverse primer is shown in SEQ ID NO:11; Preferably, the forward primer for detecting SLC34A2-ROS1_SL4:R32 is shown as SEQ ID NO:9, and the reverse primer is shown as SEQ ID NO:11; Preferably, the forward primer for detecting CD74-ROS1_C6:R32 is as shown in SEQ ID NO:10, and the reverse primer is as shown in SEQ ID NO:11; Preferably, the probes for detecting SDC4-ROS1_S2:R32, SLC34A2-ROS1_SL4:R32 and CD74-ROS1_C6:R32 are as shown in SEQ ID NO:12; Preferably, the forward primer for detecting CD74-ROS1_C6:R34 is as shown in SEQ ID NO:13, and the reverse primer is as shown in SEQ ID NO:15; Preferably, the forward primer for detecting EZR ROS_E10:R34 is shown as SEQ ID NO:14, and the reverse primer is shown as SEQ ID NO:15; Preferably, the probe for detecting CD74-ROS1_C6:R34 and EZR ROS_E10:R34 is as shown in SEQ ID NO:16; Preferably, the forward primer for detecting GOPC-ROS1_G8:R35 is shown as SEQ ID NO:17, and the reverse primer is shown as SEQ ID NO:19; Preferably, the forward primer for detecting TPM3-ROS1_T8:R35 is shown as SEQ ID NO:18, and the reverse primer is shown as SEQ ID NO:19; Preferably, the probe for detecting GOPC-ROS1_G8:R35 and TPM3-ROS1_T8:R35 is as shown in SEQ ID NO:

20.

4. The primer-probe combination for the joint detection of EML4-ALK fusion and ROS1 fusion in plasma exosomes according to any one of claims 1 to 3, It is characterized in that The primer-probe combination also includes a primer pair and a probe for detecting an internal reference gene HPRT; Preferably, the forward primer for detecting HPRT is shown as SEQ ID NO:21, and the reverse primer is shown as SEQ ID NO:23; Preferably, the probe for detecting HPRT is as shown in SEQ ID NO:

22.

5. The primer-probe combination for the joint detection of EML4-ALK fusion and ROS1 fusion in plasma exosomes according to any one of claims 1 to 4, It is characterized in that The 5' end of the probe carries a fluorescent group, and the 3' end of the probe carries a quenching group; Preferably, the fluorescent group of the probe for detecting EML4-ALK fusion and ROS1 fusion is selected from any one or a combination of at least two of FAM, VIC, TAMRA, ROX, CY5 or CY5.5; Preferably, the quenching group of the probe for detecting EML4-ALK fusion and ROS1 fusion is selected from any one or a combination of at least two of MGB, BHQ1, BHQ2, BHQ3, SQ1, SQ2 or SQ3; Preferably, the fluorescent group of the probe for detecting the internal reference gene HPRT is selected from any one of FAM, VIC, TAMRA, ROX, CY5 or CY5.5; Preferably, the quenching group of the probe for detecting the internal reference gene HPRT is selected from any one of MGB, BHQ1, BHQ2, BHQ3, SQ1, SQ2 or SQ3.

6. A kit for the combined detection of EML4-ALK fusion and ROS1 fusion in plasma exosomes, It is characterized in that The kit comprises the primer-probe combination for jointly detecting EML4-ALK fusion and ROS1 fusion in plasma exosomes according to any one of claims 1 to 5.

7. The kit for joint detection of EML4-ALK fusion and ROS1 fusion in plasma exosomes according to claim 6, It is characterized in that The kit also includes a detection reagent, which includes a reaction solution A and a reaction solution B; Preferably, the reaction solution A comprises, by molar concentration, 0.1-50 mM dATP, 0.1-50 mM dCTP, 0.1-50 mM dGTP, 0.1-100 mM dUTP, 1-500 mM MgSO 4 and 1-500 mM Tris-HCl; Preferably, the reaction solution B comprises the following components: reverse transcriptase, RNase inhibitor, hot-start DNA polymerase and thermosensitive UDG enzyme.

8. A device for combined detection of EML4-ALK fusion and ROS1 fusion in plasma exosomes, It is characterized in that The device comprises: PCR amplification module: using the primer-probe combination in the kit for joint detection of EML4-ALK fusion and ROS1 fusion in plasma exosomes according to claim 6 or 7 to prepare a PCR system, and performing PCR amplification on the sample to be tested; Result analysis module: The Ct value is obtained by fluorescence quantitative PCR detection, and EML4-ALK fusion and ROS1 fusion are judged according to the Ct value.

9. The device for jointly detecting EML4-ALK fusion and ROS1 fusion in plasma exosomes according to claim 8, It is characterized in that In the PCR system, the final concentrations of the primers for detecting EML4-ALK fusion and ROS1 fusion are independently 100-700 nM; the final concentrations of the probes are independently 25-400 nM; Preferably, the final concentrations of the primers for detecting the internal reference gene HPRT in the PCR system are independently 100-700 nM; The final concentration of the probe was 25-400 nM; Preferably, the components in the reaction solution B in the PCR system include, by final concentration, 1-1000U / 50μL reverse transcriptase, 1-500U / 50μL RNase inhibitor, 1-500U / 50μL hot start DNA polymerase and 0.1-100U / 50μL thermosensitive UDG enzyme.

10. Use of the primer-probe combination for the joint detection of EML4-ALK fusion and ROS1 fusion in plasma exosomes according to any one of claims 1 to 5 and / or the kit for the joint detection of EML4-ALK fusion and ROS1 fusion in plasma exosomes according to claim 6 or 7 in the preparation of a product for detecting lung cancer.

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