A kit and method for detecting ITK-SYK fusion genes based on ultra-high depth sequencing

Abstract

This invention provides an ITK-SYK fusion gene detection kit and method based on ultra-high-depth sequencing. The ITK-SYK fusion gene detection kit includes multiple primers. The detection method uses the obtained cf-RNA as a template to amplify the target sequence and construct a library. The obtained library is sequenced, and the sequencing data is analyzed to calculate the relative proportion of ITK-SYK fusion genes. This invention's detection method overcomes the limitations of digital PCR, designs shorter amplification fragments, further improves detection sensitivity, and uses the SE50-SE75 short read mode during sequencing, achieving ultra-high sequencing depth with minimal data volume, enabling the detection of 10... ‑6 This invention targets low-frequency fusion mutations, with the overall experimental procedure requiring as little as one day, significantly shortening the reporting cycle. It boasts high specificity, high sensitivity, simple and rapid operation, and rigorous and reliable results, providing a reliable method for research related to ITK-SYK gene fusion diseases. It is a convenient and non-invasive detection technology.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and discloses an ITK-SYK fusion gene detection kit and detection method based on ultra-high depth sequencing. Background Technology

[0002] The TEC (Tec Kinase) family is a group of non-receptor protein tyrosine kinases primarily expressed in hematopoietic cells. They play crucial roles in leukocyte development and function, participating in the regulation of various cellular processes such as cell proliferation, survival, differentiation, and cytoskeleton remodeling. Family members include BTK, ITK / TSK / EMT, TEC, TXK, and BMX, which share high structural homology. Among these, BTK and ITK are attractive drug targets. BTK can regulate Fcε receptor (FcεR) signaling in mast cells, participating in IgE-mediated diseases such as allergies, asthma, and atopic dermatitis. In T cells, ITK positively regulates the TCR signaling pathway, inducing the production of IL-2, IL-4, and IL-13. When peptide-MHC binds to homologous TCRs, ITK can be directly phosphorylated by the tyrosine protein kinase LCK, followed by autophosphorylation. ITK establishes a connection with the LAT-SLP76 complex via two SRC homologous domains, SH2 and SH3, creating a complex dependent on upstream LCK and Zap70. ITK then phosphorylates phospholipase Cγ (PLCγ), which subsequently cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to produce the second messengers inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 and DAG primarily activate NFAT and calcium signaling, targeting and activating gene promoters such as IL-2, IL-4, and IL-13.

[0003] Gene fusion refers to the abnormal connection of two originally independent genes on a chromosome, forming a new fused gene. Gene fusion is particularly important in cancer research because it can lead to abnormal activation of intracellular signaling pathways, thereby promoting tumor development and progression. Gene fusion generally occurs at the genomic level. Depending on the location of the fusion breakpoint, some gene fusions involve transcription while others do not. Besides genomic fusion, gene fusion can also occur at the transcriptomic level. Peripheral blood T-cell lymphomas (PTCLs) are among the most aggressive non-Hodgkin lymphomas, with high mortality rates and low efficacy of conventional chemotherapy. The chromosomal translocation t(5;9)(q33;q22) induces interleukin-2 (IL-2) to induce T-cell kinase (ITK)-spleen tyrosine kinase (SYK) fusion, which has been identified as a relapse event in PTCL. Identifying and characterizing kinase fusions in human malignant tumors is crucial for identifying oncological targets; therefore, it is essential to detect and study ITK-SYK gene fusions.

[0004] With technological advancements, common molecular pathological methods for gene fusion detection now include: fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), digital PCR, and next-generation sequencing (NGS). FISH is complex, difficult to perform, and has low sensitivity, and is used to identify gene fusions at the chromosomal level. RT-PCR can detect fusion transcripts with high sensitivity at the RNA level; however, this method is significantly affected by RNA quality, potentially leading to false negatives or false positives. IHC is more economical and faster than current molecular detection methods, but result interpretation is somewhat subjective, and weakly positive results require further verification using techniques like FISH. Due to limitations in amplified fragment length, digital PCR has limited sensitivity, capable of detecting only 10-1 gene fusions. -4 The above mutation frequencies, coupled with the specificity issues of fluorescent probes, may lead to false negatives or false positives. NGS is divided into targeted capture and multiplex amplification. Targeted capture has limitations such as high system cost, complex experimental operation, and long overnight hybridization time. Summary of the Invention

[0005] This invention addresses the aforementioned deficiencies in existing technologies by providing an ITK-SYK fusion gene detection kit and method based on ultra-high-depth sequencing. The detection method of this invention can effectively detect low-frequency mutations of 0.0001% in the ITK-SYK fusion gene, exhibiting high specificity, high sensitivity, ease of operation, and rigorous and reliable results, thus providing a reliable method for research related to ITK-SYK gene fusion diseases.

[0006] The technical solution of the present invention is as follows:

[0007] This invention provides an ITK-SYK fusion gene detection kit based on ultra-high depth sequencing, comprising a primer amplification system and a library amplification system; the primer amplification system comprises a specific primer mix of nucleotides as shown in SEQ ID NO.1 to SEQ ID NO.5; the library amplification system comprises a library amplification primer mix composed of an upstream primer selected from any of the nucleotides shown in SEQ ID NO.6 to SEQ ID NO.13 and a downstream primer selected from any of the nucleotides shown in SEQ ID NO.14 to SEQ ID NO.25.

[0008] This invention also provides a method for detecting ITK-SYK fusion genes based on ultra-high depth sequencing, comprising the following steps:

[0009] S1: Extract cf-RNA from plasma and reverse transcribe it into cDNA;

[0010] S2: The cDNA is amplified using the primer amplification system described above to obtain the first round of primer amplification and the first round of product.

[0011] S3: The first-round product is used to perform a second-round library amplification using the above-described library amplification system to obtain the second-round product;

[0012] S4: The second-round product was purified using magnetic beads. After library construction, the library concentration and fragment length were measured and recorded to prepare for sequencing.

[0013] S5: The obtained library was subjected to short-read ultra-high-depth sequencing using a sequencing platform;

[0014] S6: After the data is processed, it is compared with the reference sequence to analyze whether gene fusion has occurred in the sample and the relative expression level of gene fusion.

[0015] Furthermore, the primer amplification system in S2 is 25 μL, including 12.5 μL 2×Multiplex PCR Buffer, 2 μL specific primer Mix (5 μm), 1 μL Taq Pro Multiplex DNA Polymerase, and 9.5 μL cDNA.

[0016] Furthermore, the composition of the specific primer Mix is ​​shown in the table below:

[0017] Serial Number Primer name Volume of 100μm stock solution added (μL) SEQ ID NO.1 SYK_F 5 SEQ ID NO.2 ITK_R1 5 SEQ ID NO.3 ITK_R2 5 SEQ ID NO.4 RPP_F 5 SEQ ID NO.5 RPP_R 5 margin <![CDATA[ddH2O]]> 75 .

[0018] Furthermore, the first round of primer amplification reaction conditions in S2 include: i) holding at 95°C for 5 minutes; ii) holding at 95°C for 30 seconds, then at 63°C for 1 minute, and finally at 72°C for 20 seconds; this step is repeated 30 times; iii) holding at 72°C for 10 minutes; and iiii) holding at 12°C.

[0019] Furthermore, the library amplification system in S3 is 25 μL, including 12.5 μL 2×PCR Buffer, 2 μL library amplification primer Mix (5 μm), 0.5 μL high-fidelity Taq enzyme, 2 μL first-round product, and 8 μL ddH2O.

[0020] Furthermore, the composition of the library amplification primer Mix is ​​shown in the table below:

[0021] Serial Number Primer name Volume of 100μm stock solution added (μL) SEQ ID NO.6 to SEQ ID NO.13 I5 primer 5 SEQ ID NO.14 to SEQ ID NO.25 I7 primer 5 margin <![CDATA[ddH2O]]> 90 .

[0022] Furthermore, the second round of library amplification reaction conditions in S3 include: i) holding at 98°C for 3 minutes; ii) holding at 98°C for 10 seconds, then at 60°C for 30 seconds, and finally at 72°C for 20 seconds; this step is repeated 10 times; iii) holding at 72°C for 5 minutes; iiii) holding at 12°C.

[0023] The outstanding effects of this invention are:

[0024] This invention proposes an ITK-SYK fusion gene detection kit and method based on ultra-high-depth sequencing. The ITK-SYK fusion gene detection kit includes multiple primers. The detection method involves extracting cf-RNA from plasma, reverse transcribing it into cDNA, and then constructing a library by multiplex amplification of the ITK-SYK fusion gene and an internal reference gene fragment. The resulting library is then subjected to short-read ultra-high-depth sequencing. Finally, the data is compared with a reference sequence to analyze whether gene fusion has occurred in the sample and the relative expression level of the gene fusion. This invention's detection method overcomes the limitations of digital PCR, designing shorter amplification fragments to further improve detection sensitivity. Sequencing uses the SE50-SE75 short-read mode, achieving ultra-high sequencing depth with minimal data volume, enabling the detection of 10... -6 This invention targets low-frequency fusion mutations, with the overall experimental procedure requiring as little as one day, significantly shortening the reporting cycle. It boasts high specificity, high sensitivity, simple and rapid operation, and rigorous and reliable results, providing a reliable method for research related to ITK-SYK gene fusion diseases. It is a convenient and non-invasive detection technology.

[0025] The following detailed description of specific embodiments of the present invention will make the technical solution of the present invention easier to understand and master. Attached Figure Description

[0026] Figure 1 This is a flowchart illustrating the detection process of the ITK-SYK fusion gene in an embodiment of the present invention.

[0027] Figure 2 This is a schematic diagram of the amplification process according to an embodiment of the present invention;

[0028] Figure 3 This is a graph showing the results of verifying the specificity of an embodiment of the present invention;

[0029] Figure 4 The graph shows the results of verifying the detection limit in an embodiment of the present invention. Detailed Implementation

[0030] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0031] Example:

[0032] like Figure 1 As shown, the steps of an ITK-SYK fusion gene detection method based on ultra-high depth sequencing in this embodiment are as follows:

[0033] 1. Plasma cf-RNA extraction

[0034] cf-RNA extraction was performed using the Quick-cfRNA Serum & Plasma Kit (ZYMO RESEARCH). >50 μL of plasma sample was centrifuged at ≥12000×g for 15 min to remove cell debris and precipitate.

[0035] Take a new 15mL centrifuge tube and add 200μL of Quick-cfRNA for every 200μL of sample. TM Mix well with Digestion Buffer. (If the sample volume is <200 μL, make up to 200 μL with 1×PBS; if the sample volume is ≥1.5 mL, use a 50 mL centrifuge tube for subsequent operations.)

[0036] Add 10 μL of Proteinase K to every 200 μL of sample and vortex mix for 10 seconds.

[0037] Incubate at 37°C for 2 hours.

[0038] Add 1 volume of Quick-cfRNA TM Add the binding buffer to the mixture from the previous digestion step and vortex for 10 seconds.

[0039] Add 1.5 times the volume of 100% isopropanol to the mixture from the previous step and vortex mix for 10 seconds.

[0040] Fit the 25mL funnel onto the Spin-Away TM In the filter (yellow purification column), after tight coupling, it is loaded onto a negative pressure multi-connector.

[0041] Pour all the mixture into a 25mL funnel and turn on the vacuum switch to allow the liquid to pass completely through the Spin-Away. TM Filter purification column.

[0042] After confirming that the liquid has completely passed through and there is no residue, turn off the vacuum switch and disconnect the Spin-Away valve. TM A tightly connected 25mL funnel for filtering. (Spin-Away) TM(The filter is still on the negative pressure multi-connector)

[0043] To Spin-Away TM Add 600 μL of RNA Prep Buffer to the filter, turn on the vacuum switch, allow the liquid to pass through completely, and then turn off the vacuum switch.

[0044] Spin-Away TM Remove the filter and transfer it to a new 2mL collection tube. Centrifuge for 2 minutes to remove any residue, then transfer it to a new 1.5mL centrifuge tube.

[0045] To Spin-Away TM Add 200 μL of RNA Recovery Buffer to the filter, let it stand at room temperature for 3 min, centrifuge, and retain the filtrate in 1.5 mL centrifuge tube.

[0046] Add 300 μL of ethanol (95-100%) to the filtrate, mix well and centrifuge briefly.

[0047] Zymo-Spin TM Attach the IC column to a new 2mL centrifuge tube, pour in all the mixture, centrifuge, and discard the filtrate.

[0048] To Zymo-Spin TM Add 400 μL of RNA Prep Buffer to the IC Column, centrifuge, and discard the filtrate.

[0049] Add 700 μL RNA Wash Buffer, centrifuge, and discard the filtrate.

[0050] Add 400 μL RNA Wash Buffer and centrifuge for 2 min; remove residue and then transfer to a new 1.5 mL centrifuge tube.

[0051] To Zymo-Spin TM Add 15 μL of DNase / RNase-Free Water to the IC column, let stand at room temperature for 2 min, centrifuge, and retain the filtrate in the centrifuge tube for 1.5 mL.

[0052] 2. Genomic DNA removal

[0053] Prepare the reaction solution in RNase-free centrifuge tubes according to Table 1:

[0054] Table 1 Reaction Solution Preparation Table

[0055] reagents μL / test <![CDATA[RNase-free ddH2O]]> to 10 5×gDNA wiper Mix 2 Total RNA or Poly A+ RNA 8 Total 10

[0056] Gently mix with a pipette and incubate at 42°C for 2 minutes.

[0057] 3. Prepare the first-strand cDNA synthesis reaction solution

[0058] Prepare the reaction solution in the RNase-free PCR tube according to Table 2:

[0059] Table 2 Reaction Solution Preparation Table

[0060] reagents μL / test 10×RT Mix 2 HiScript III Enzyme Mix 2 Oligo(dT)20VN 1 Random hexamers 1 <![CDATA[RNase-free ddH2O]]> 4 Total 10

[0061] Gently mix the mixture with a pipette, add the prepared reaction solution mix to the mixture from the previous step, and perform the PCR reaction according to the reaction conditions in Table 3:

[0062] Table 3 PCR reaction conditions

[0063] Temperature (°C) time 37 15min 85 5 seconds

[0064] The first-strand cDNA product can be used immediately; or stored at -20°C for a short period.

[0065] 4. Specific PCR reaction

[0066] Prepare primer mixes according to Table 4 (sequences are shown in Table 10), with a primer working solution concentration of 5 μM:

[0067] Table 4 Primer Mix Configuration Table

[0068]

[0069] Prepare the reaction solution according to Table 5:

[0070] Table 5 Reaction Solution Preparation Table

[0071] system μL / test 2×Multiplex PCR Buffer 12.5 Primer Mix (5μm) 2 Taq Pro Multiplex DNA Polymerase 1 cDNA 9.5 MIX size 25

[0072] Close the hot cap and run the reaction program as shown in Table 6 below:

[0073] Table 6 PCR reaction system

[0074]

[0075] 5. Index PCR

[0076] Prepare primer mixes according to Table 7 (sequences are shown in Table 10), with a primer working solution concentration of 5 μM:

[0077] Table 7 Primer Mix Configuration Table

[0078]

[0079]

[0080] Prepare the reaction solution according to Table 8:

[0081] Table 8 Reaction Solution Preparation Table

[0082] Components μL / test 2×PCR buffer 12.5 I5 / I7 primer (5μm) 2 High-fidelity Taq enzyme 0.5 First-round products 2 <![CDATA[ddH2O]]> 8 MIX size 25

[0083] Close the hot cap and run the reaction program as shown in Table 9 below:

[0084] Table 9 PCR Reaction System

[0085]

[0086] 6. Segment Sorting

[0087] Remove the magnetic bead solution from 2-8℃ 30 minutes in advance, let it stand to allow its temperature to equalize to room temperature, and then invert or vortex to mix the magnetic bead solution thoroughly.

[0088] Two different ratios of magnetic beads (50 μL and 15 μL) were added to the eight-unit tube and labeled accordingly.

[0089] Take 25 μL of the secondary PCR product, add 25 μL of water to make up to 50 μL, mix well, and then transfer the entire amount to 50 μL of labeled magnetic beads.

[0090] Mix thoroughly and incubate at room temperature for 5 minutes to allow the product to bind to the magnetic beads.

[0091] Place the sample on a magnetic rack and wait for the solution to clarify (about 3 minutes). Then, aspirate the supernatant into 15 μL of magnetic beads.

[0092] Mix thoroughly and incubate at room temperature for 5 minutes to allow the product to bind to the magnetic beads.

[0093] Place the sample on the magnetic rack and wait for the solution to clarify (about 3 minutes), then carefully remove the supernatant.

[0094] Keep the sample on the magnetic rack at all times, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant.

[0095] Rinse once more.

[0096] After a brief centrifugation, place the sample on a magnetic rack and wait for the magnetic beads to leave the liquid surface. Remove the remaining ethanol from the eight-panel container and dry the magnetic beads at room temperature until they become matte.

[0097] Remove the sample from the magnetic rack, add 22.5 μL of nuclease-free water, vortex or pipette to mix thoroughly, and let stand at room temperature for 2 minutes.

[0098] After allowing the samples to clarify on a magnetic rack, carefully aspirate 20 μL of the supernatant into a 1.5 mL centrifuge tube corresponding to each library.

[0099] The concentration of the library was determined using a Qubit 4.0 fluorometer, and the normal concentration range should be 5-20 ng / μL.

[0100] The bands were confirmed using 1.2% agarose gel electrophoresis. A normal band should be around 180 bp.

[0101] 7. Sequencing

[0102] Using a 2200 Tapestation (Agilent Technologies) to perform fragment quality checks on the library, the main peak should be around 180bp.

[0103] The high-throughput gene sequencer used for sequencing was a FASTASeq300 (Shenzhen Zhenmai Biotechnology Co., Ltd.), and the sequencing mode was SE50. (If 10...) -5 For detection sensitivity, >1Mreads need to be measured; if 10Mreads are required... -6 For detection sensitivity, >10M reads need to be measured.

[0104] Table 10 Amplification Primer Sequences

[0105]

[0106]

[0107] 8. Test Results

[0108] like Figure 2 As shown, the results analysis used the ITK-SYK fusion fragment divided by the Rpp30 gene fragment as the relative quantitative value. Since a nested PCR reaction system was used, the Rpp30 fragment was calculated using the formula (ITK-SYK fusion long fragment + ITK-SYK fusion short fragment) / 2.

[0109] The specific results are as follows:

[0110] (1) Specificity test

[0111] One sample each of ITK-SYK positive cell line supernatant (P), ITK-SYK negative cell line supernatant (N), and healthy human plasma (H) was selected. The primers were separated for testing, and the results are as follows: Figure 3 As shown in Table 11, positive bands were detected in both SF1-IR1 (fusion long fragment) and SF1-IR2 (fusion short fragment) of P sample, while no positive bands were detected in N and H samples.

[0112] Table 11 Specificity Test Results

[0113] Excerpt Fragment size (bp) P N H SF1-IR1 100 + - - SF1-IR2 87 + - -

[0114] Table 11 shows that the ITK-SYK fragment was detectable in the supernatant of positive cell lines, while it was absent in the supernatant of negative cell lines and normal human plasma. This demonstrates that: 1. Cell supernatant contains sufficient amounts of recognizable cfRNA; 2. The SF1-IR2 primers and SF1-IR2 primer set included in this kit can specifically amplify the ITK-SYK fragment; 3. The amplified fragment is only present in positive samples; thus confirming the effectiveness and specificity of this method.

[0115] (2) Detection limit test

[0116] Supernatant from ITK-SYK fusion gene-positive cells was selected, and serial dilutions of the positive cell supernatant were performed using negative cell supernatant at ratios of 1:10, 1:100, 1:1000, 1:10000, 1:100000, and 1:1000000 for testing. Results are as follows: Figure 4 As shown in Table 12.

[0117] Table 12 Detection Limit Test Results

[0118] Excerpt 1:10 1:100 1:1000 1:10000 1:100000 1:1000000 Fusion fragments 56038 35402 19653 1097 440 172 Internal reference fragments 258214 302188 339956 183881 168924 110653 relative expression level 0.1085 0.0586 0.0289 0.0030 0.0013 0.0008

[0119] Table 12 shows that ITK-SYK fusion slices can still be detected even at a 1:1,000,000 dilution of the cell supernatant. This demonstrates the extremely high sensitivity of this method.

[0120] (3) Testing of PTCL animal model induced by ITK-SYK fusion gene

[0121] Three ITK-SYK fusion gene-induced PTCL mice (n=3) and three control mice were selected. Blood was collected from the tail vein of the mice, and 50 μL of plasma was separated for testing. The results are shown in Table 13.

[0122] Table 13 PTCL Animal Model Test Results

[0123] Excerpt Control mouse 1 Control mouse 2 Control mouse 3 1 diseased mouse 2 infected mice 3 infected rats Fusion fragments 0 0 0 216 101 518 Internal reference fragments 12635 9396 8470 4471 8353 7497 relative expression level 0.000％ 0.000％ 0.000％ 2.416％ 0.605％ 3.455％

[0124] Table 13 shows that the ITK-SYK fragment could not be detected in the serum of control mice using this method, while the fusion fragment was specifically detected in the plasma of diseased mice. This demonstrates the high specificity of this method.

[0125] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. An ultra-deep sequencing-based ITK-SYK fusion gene detection kit, characterized in that, The primer amplification system comprises a specific primer Mix as shown in nucleotides of SEQ ID NO. 1 to SEQ ID NO. 5; and the library amplification system comprises a library amplification primer Mix consisting of an upstream primer as shown in nucleotides of SEQ ID NO. 6 to SEQ ID NO. 13 and a downstream primer as shown in nucleotides of SEQ ID NO. 14 to SEQ ID NO.

25. The primer amplification system comprises a specific primer Mix as shown in nucleotides of SEQ ID NO. 1 to SEQ ID NO. 5; and the library amplification system comprises a library amplification primer Mix consisting of an upstream primer as shown in nucleotides of SEQ ID NO. 6 to SEQ ID NO. 13 and a downstream primer as shown in nucleotides of SEQ ID NO. 14 to SEQ ID NO. 25.

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