Ultrahigh depth sequencing-based ITK-SYK fusion gene detection kit and detection method

Abstract

The invention provides an ITK-SYK fusion gene detection kit and detection method based on ultra-high depth sequencing, the ITK-SYK fusion gene detection kit comprises a plurality of primers, the detection method uses obtained cf-RNA as a template to amplify a target sequence and build a library, the obtained library is sequenced, offline data is analyzed, and the detection result of the ITK-SYK fusion gene detection kit based on ultra-high depth sequencing is obtained. And calculating the relative proportion of the ITK-SYK fusion gene. According to the detection method, the limit of digital PCR is broken through, the designed amplified fragment is shorter, the detection sensitivity is further improved, an SE50-SE75 short read length mode is used during sequencing, ultrahigh sequencing depth is achieved under the condition of extremely small data volume, 10 <-6 > low-frequency fusion mutation can be detected, the time required by the overall experimental process is as short as 1 day, and the report period is greatly shortened. The method is high in specificity, high in sensitivity, simple, convenient and rapid to operate and rigorous and reliable in result, provides a reliable method for related research of ITK-SYK gene fusion diseases, and is a convenient and non-invasive detection technology.

Description

Technical Field

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

[0002] The TEC (Tec Kinase) family is a group of non-receptor protein tyrosine kinases primarily expressed in hematopoietic cells. They play a crucial role in the development and function of leukocytes, regulating a variety of cellular processes, including cell proliferation, survival, differentiation, and cytoskeletal reorganization. Family members include BTK, ITK / TSK / EMT, TEC, TXK, and BMX, which share a high degree of structural homology. BTK and ITK are attractive drug targets. BTK regulates Fcε receptor (FcεR) signaling in mast cells and is implicated 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. Upon peptide-MHC binding to the cognate TCR, ITK is directly phosphorylated by the tyrosine protein kinase LCK and subsequently undergoes autophosphorylation. ITK establishes a connection with the LAT-SLP76 complex through two SRC homology domains, SH2 and SH3, creating a complex that is dependent on the upstream LCK and Zap70. ITK then phosphorylates phospholipase Cγ (PLCγ), which then 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 gene promoters for activation, such as IL-2, IL-4, and IL-13.

[0003] Gene fusion refers to the abnormal connection of two previously independent genes on a chromosome, forming a new fusion 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 fusions generally occur at the genomic level. Depending on the location of the fusion breakpoint, some gene fusions are transcribed while others are not. In addition to genomic fusions, gene fusions can also occur at the transcript level. Peripheral T-cell lymphomas (PTCLs) are among the most aggressive non-Hodgkin lymphomas, with high mortality and low efficacy of conventional chemotherapy. The chromosomal translocation t(5;9)(q33;q22) that produces interleukin-2 (IL-2)-induced T-cell kinase (ITK)-spleen tyrosine kinase (SYK) fusion tyrosine kinase has been identified as a recurrent event in PTCL. The identification and characterization of kinase fusions in human malignancies is an important approach to identifying oncological targets. Therefore, the detection and study of ITK-SYK gene fusions are highly warranted.

[0004] With the advancement of technology, the common molecular pathology detection methods for gene fusion detection currently include: fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), digital PCR, NGS, etc. FISH operation is complex, difficult, and has low sensitivity. It is used to identify gene fusions at the chromosomal level. RT-PCR can detect fusion transcripts at the RNA level with high sensitivity, but this method is greatly affected by the quality of RNA, so it may cause some false negative or false positive results. Compared with the current existing molecular detection methods, IHC is more economical and faster, but there is a certain degree of subjectivity in the interpretation of the results. The judgment of weak positive results needs to be further verified by FISH and other technologies. Limited by the length of the amplified fragment, the sensitivity of digital PCR is limited, and it can only detect 10 -4 The mutation frequency above this threshold, coupled with specificity issues with fluorescent probes, can lead to false negatives or positives. NGS is divided into targeted capture and multiplex amplification. Targeted capture has limitations such as high system cost, complex experimental procedures, and the long overnight hybridization time required. Summary of the Invention

[0005] The present invention addresses the aforementioned deficiencies in the prior art by providing an ITK-SYK fusion gene detection kit and method based on ultra-deep sequencing. The method can effectively detect low-frequency mutations in the ITK-SYK fusion gene as low as 0.0001%, demonstrating strong specificity, high sensitivity, ease of use, and reliable results. This method provides a robust approach for research related to ITK-SYK gene fusion diseases.

[0006] The technical solutions of the present invention are as follows:

[0007] The present 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 such as SEQ ID NO.1 to SEQ ID NO.5; the library amplification system comprises a library amplification primer mix composed of any upstream primer selected from nucleotides such as SEQ ID NO.6 to SEQ ID NO.13 and any downstream primer selected from nucleotides such as SEQ ID NO.14 to SEQ ID NO.25.

[0008] The present 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 convert it into cDNA through reverse transcription;

[0010] S2: The cDNA is subjected to a first round of primer amplification using the primer amplification system of claim 1 to obtain a first round product;

[0011] S3: The first round product is subjected to a second round of library amplification using the library amplification system of claim 1 to obtain a second round product;

[0012] S4: Purify the second-round product using magnetic beads. After library construction is completed, measure and record the library concentration and fragment length to prepare for sequencing.

[0013] S5: Use a sequencing platform to perform short-read ultra-high-depth sequencing on the obtained library;

[0014] S6: After the data is downloaded from the machine, it is compared with the reference sequence to analyze whether gene fusion occurs 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 following table:

[0017] Serial number Primer name 100μm stock solution input volume (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 reaction conditions for the first round of primer amplification in S2 include: i) maintaining at 95°C for 5 minutes; ii) maintaining at 95°C for 30 seconds, then maintaining at 63°C for 1 minute, and finally maintaining at 72°C for 20 seconds; this step is repeated 30 times; iii) maintaining at 72°C for 10 minutes; iiii) maintaining 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 following table:

[0021] Serial number Primer name 100μm stock solution input volume (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 reaction conditions for the second round of library amplification in S3 include: i) maintaining at 98°C for 3 minutes; ii) maintaining at 98°C for 10 seconds, then maintaining at 60°C for 30 seconds, and finally maintaining at 72°C for 20 seconds; this step is repeated 10 times; iii) maintaining at 72°C for 5 minutes; iiii) maintaining at 12°C.

[0023] The outstanding effects of the present invention are:

[0024] The present invention proposes an ITK-SYK fusion gene detection kit and detection method based on ultra-high depth sequencing. The ITK-SYK fusion gene detection kit includes multiple primers. The detection method extracts cf-RNA from plasma, reverse transcribes it into cDNA, and then constructs a library by multiple amplification of ITK-SYK fusion gene and internal reference gene fragments. The resulting library is then subjected to short-read ultra-high depth sequencing. Finally, the data is compared with the reference sequence after being downloaded to analyze whether the sample has gene fusion and the relative expression level of the gene fusion. The detection method of the present invention breaks through the limits of digital PCR, designs shorter amplified fragments, further improves detection sensitivity, uses SE50-SE75 short read length mode for sequencing, has ultra-high sequencing depth with extremely small data volume, and can detect 10 -6 The present invention can detect low-frequency fusion mutations in patients with ITK-SYK gene fusions, with the overall experimental process taking as little as one day, significantly shortening the reporting cycle. The present invention has strong specificity, high sensitivity, simple and rapid operation, and rigorous and reliable results. It provides a reliable method for the study of diseases related to ITK-SYK gene fusions and is a convenient, non-invasive detection technology.

[0025] The specific implementation methods of the present invention are further described below in conjunction with embodiments to make the technical solutions of the present invention easier to understand and grasp. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Flow chart of the detection of ITK-SYK fusion gene in an embodiment of the present invention;

[0027] Figure 2 Schematic diagram of amplification according to an embodiment of the present invention;

[0028] Figure 3 This is a result diagram for verifying the specificity of the embodiment of the present invention;

[0029] Figure 4 This is a result diagram for verifying the lower limit of detection in an embodiment of the present invention. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0031] Example:

[0032] like Figure 1 As shown, the steps of the 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 was extracted using the Quick-cfRNA Serum & Plasma Kit (ZYMO RESEARCH). >50 μL of sample plasma was centrifuged at a centrifugal force of ≥12,000 × g for 15 min to remove cell debris and precipitate.

[0035] Take a new 15mL centrifuge tube and add 200μL Quick-cfRNA for every 200μL of sample. TM Mix the Digestion Buffer. (If the added sample volume is <200 μL, use 1×PBS to make up to 200 μL. If the added 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 Binding Buffer to the digestion mixture in the previous step and vortex to mix 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] Put the 25mL funnel onto the Spin-Away TM Filter (yellow purification column), tightly connect and load onto the negative pressure manifold.

[0041] Pour all the mixture into the 25mL funnel and turn on the vacuum switch to allow the liquid to completely pass 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 unplug the Spin-Away TM Filter tightly connected 25mL funnel. (Spin-Away TMFilter is still on the negative pressure manifold)

[0043] Spin-Away TM Add 600 μL RNAPrep 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; remove the residue and transfer it to a new 1.5mL centrifuge tube.

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

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

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

[0048] To Zymo-Spin TM Add 400 μL 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 minutes; remove the residue and 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 it stand at room temperature for 2 minutes, and retain the filtrate in a 1.5 mL centrifuge tube after centrifugation.

[0052] 2. Genomic DNA Removal

[0053] In an RNase-free centrifuge tube, prepare the reaction solution according to Table 1:

[0054] Table 1 Reaction solution configuration 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] Mix gently by pipetting and incubate at 42°C for 2 min.

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

[0058] In an RNase-free PCR tube, prepare the reaction mixture according to Table 2:

[0059] Table 2 Reaction solution configuration 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] Use a pipette to gently pipette to mix, add the prepared reaction solution mix to the mixture after the previous step, and perform PCR reaction according to the reaction conditions in Table 3:

[0062] Table 3 PCR reaction conditions

[0063] Temperature (℃) time 37 15min 85 5sec

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

[0065] 4. Specific PCR reaction

[0066] Prepare primer mix 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] Serial number Primer name 100μm stock solution input volume (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

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

[0070] Table 5 Reaction solution configuration table

[0071]

[0072]

[0073] Close the heated lid and run the reaction program as shown in Table 6:

[0074] Table 6 PCR reaction system

[0075]

[0076] 5. Index PCR

[0077] Prepare primer mix according to Table 7 (sequences are shown in Table 10), with a primer working solution concentration of 5 μM:

[0078] Table 7 Primer Mix Configuration Table

[0079] Serial number Primer name 100μm stock solution input volume (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

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

[0081] Table 8 Reaction solution configuration table

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

[0083] Close the heated lid and run the reaction program as shown in Table 9:

[0084] Table 9 PCR reaction system

[0085]

[0086] 6. Fragment sorting

[0087] Take out the magnetic bead solution from 2-8°C 30 minutes in advance, let it stand to balance its temperature to room temperature, and invert or vortex to mix the magnetic bead solution thoroughly.

[0088] Add two ratios of magnetic beads (50 μL and 15 μL) to eight tubes and mark them.

[0089] Pipette all 25 μL of the secondary PCR product, add 25 μL of water to make up the volume to 50 μL, mix well and transfer all to the labeled 50 μL 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 stand, wait for the solution to become clear (approximately 3 minutes), and then pipette 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 a magnetic rack, wait for the solution to become clear (approximately 3 minutes), and carefully remove the supernatant.

[0094] Keep the sample on the magnetic stand, 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] Repeat the rinse cycle.

[0096] Centrifuge briefly and place the sample on a magnetic rack. Wait until the magnetic beads leave the liquid surface, aspirate the remaining ethanol in the eight strips, and dry the magnetic beads at room temperature with the lid open until matte.

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

[0098] After the sample was allowed to stand on the magnetic stand to clarify, 20 μL of the supernatant was carefully pipetted into the 1.5 mL centrifuge tube corresponding to each library.

[0099] The concentration of the library was measured using a Qubit 4.0 fluorometer. The normal concentration range was 5-20 ng / μL.

[0100] Band confirmation was performed using 1.2% agarose gel electrophoresis; the normal band should be around 180 bp.

[0101] 7. Sequencing

[0102] The library was quality checked using 2200tapestation (Agilent Technologies, Inc.), and the main peak should be around 180 bp.

[0103] The high-throughput gene sequencer used for sequencing was FASTASeq300 (Shenzhen Zhenmai Biotechnology Co., Ltd.), and the sequencing mode was SE50. -5 Detection sensitivity requires >1M reads. If 10 -6 Detection sensitivity requires measuring >10M reads.

[0104] Table 10 Amplification primer sequences

[0105]

[0106]

[0107] 8. Test results

[0108] like Figure 2 As shown, the result 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 formula used was (ITK-SYK fusion long fragment + ITK-SYK fusion short fragment) / 2 times the Rpp30 fragment.

[0109] The specific results are as follows:

[0110] (1) Specificity test

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

[0112] Table 11 Specificity test results

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

[0114] Table 11 shows that ITK-SYK fragments were detected in the supernatants of positive cell lines, but absent in the supernatants of negative cell lines and normal human plasma. This demonstrates that: 1. the cell supernatants contain sufficient amounts of identifiable cfRNA; 2. the SF1-IR2 primers and SF1-IR2 primer set included in this kit can specifically amplify ITK-SYK fragments; and 3. the amplified fragments are present only in positive samples, confirming the effectiveness and specificity of this method.

[0115] (2) Detection limit test

[0116] The supernatant of ITK-SYK fusion gene positive cells was selected and the supernatant of negative cells was used to perform gradient dilution of the positive cell supernatant, and the dilutions were 1:10, 1:100, 1:1000, 1:10000, 1:100000, and 1:1000000 for testing. Figure 4 and as shown in Table 12.

[0117] Table 12 Detection limit test results

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

[0119] Table 12 shows that ITK-SYK fusion fragments can still be detected at a dilution of 1:1,000,000 in the cell supernatant, demonstrating the high sensitivity of this method.

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

[0121] Three mice with PTCL induced by the ITK-SYK fusion gene 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] snippet Control rat 1 Control rat 2 Control rat 3 Diseased rat 1 Diseased rat 2 Diseased rat 3 Fusion fragment 0 0 0 216 101 518 Internal reference fragment 12635 9396 8470 4471 8353 7497 Relative expression 0.000％ 0.000％ 0.000％ 2.416％ 0.605％ 3.455％

[0124] Table 13 shows that the present method could not detect the ITK-SYK fragment in the serum of control mice, but specifically detected the fusion fragment in the plasma of diseased mice. This shows that the present method has high specificity.

[0125] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A kit for detecting ITK-SYK fusion genes based on ultra-high depth sequencing, characterized in that: It includes a primer amplification system and a library amplification system; the primer amplification system includes a specific primer Mix of nucleotides as shown in SEQ ID NO.1 to SEQ ID NO.5; the library amplification system includes a library amplification primer Mix composed of any upstream primer selected from nucleotides as shown in SEQ ID NO.6 to SEQ ID NO.13 and any downstream primer selected from nucleotides as shown in SEQ ID NO.14 to SEQ ID NO.

25.

2. A method for detecting ITK-SYK fusion gene based on ultra-high depth sequencing, characterized in that: The following steps are involved: S1: Extract cf-RNA from plasma and convert it into cDNA through reverse transcription; S2: The cDNA is subjected to a first round of primer amplification using the primer amplification system of claim 1 to obtain a first round product; S3: The first round product is subjected to a second round of library amplification using the library amplification system of claim 1 to obtain a second round product; S4: Purify the second-round product using magnetic beads. After library construction is completed, measure and record the library concentration and fragment length to prepare for sequencing. S5: Use a sequencing platform to perform short-read ultra-high-depth sequencing on the obtained library; S6: After the data is downloaded from the machine, it is compared with the reference sequence to analyze whether gene fusion occurs in the sample and the relative expression level of gene fusion.

3. The detection method according to claim 2, characterized in that 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.

4. The detection method according to claim 3, characterized in that The composition of the specific primer Mix is ​​shown in the following table: 。 5. The detection method according to claim 2, characterized in that The reaction conditions for the first round of primer amplification in S2 include: i) maintaining at 95°C for 5 minutes; ii) maintaining at 95°C for 30 seconds, then maintaining at 63°C for 1 minute, and finally maintaining at 72°C for 20 seconds; this step is repeated 30 times; iii) maintaining at 72°C for 10 minutes; iiii) maintaining at 12°C.

6. The detection method according to claim 2, characterized in that 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.

7. The detection method according to claim 6, characterized in that The composition of the library amplification primer Mix is ​​shown in the following table:

8. The detection method according to claim 2, characterized in that The reaction conditions for the second round of library amplification in S3 include: i) maintaining at 98°C for 3 minutes; ii) maintaining at 98°C for 10 seconds, then maintaining at 60°C for 30 seconds, and finally maintaining at 72°C for 20 seconds; this step is repeated 10 times; iii) maintaining at 72°C for 5 minutes; iiii) maintaining at 12°C.

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