Probe composition for detecting synovial sarcoma SS18-SSX fusion gene as well as preparation method and application of probe composition
By designing a four-color fluorescent in situ hybridization probe composition, the problem of distinguishing SS18-SSX fusion gene types in synovial sarcoma was solved, enabling rapid and accurate diagnosis and prognosis, while reducing costs and complexity.
Patent Information
- Application Number
- CN202411432172.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-14
- Publication Date
- 2025-02-18
AI Technical Summary
Existing technologies are insufficient to quickly and accurately distinguish different SS18-SSX fusion gene types in synovial sarcoma. Traditional methods are time-consuming and labor-intensive, and it is difficult to distinguish between SSX1 and SSX4 fusion types.
A four-color fluorescent in situ hybridization probe composition was designed, including the 5' and 3' probe sets of the SS18 gene and the 3' probe sets of the SSX1 and SSX4 genes. Each probe set was labeled with a different color fluorescent marker to cover a specific gene region. The SS18-SSX1, SS18-SSX2 and SS18-SSX4 fusions were distinguished by the signal type.
This technology enables one-step differentiation of the three fusion types SS18-SSX at the FISH level, eliminating the need for a second probe design, reducing preparation costs and hybridization background, improving signal-to-noise ratio and specificity, and simplifying the diagnostic process.
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Abstract
Description
Technical Field
[0001] The present application belongs to the field of molecular biology, and specifically relates to a probe composition for detecting the SS18-SSX fusion gene of synovial sarcoma, and a preparation method and application thereof. Background Art
[0002] Synovial sarcoma (SS) is a highly malignant soft tissue sarcoma, accounting for about 5%-10% of soft tissue sarcomas. Synovial sarcoma often affects the limbs, but can also occur in any other part of the body, such as the lungs, liver, peritoneum, kidneys, etc. Histologically, synovial sarcoma has a high degree of heterogeneity, which makes the diagnosis of synovial sarcoma very difficult, especially the diagnosis of puncture tissue or small biopsy specimens is challenging. In molecular genetics, synovial sarcoma has a characteristic chromosomal translocation t(X; 18)(p11.2; q11.2), which causes the fusion of the SS18 gene at 18q11.2 and the SSX gene family (SSX1, SSX2, SXX4) at Xp11.2, resulting in the SS18-SSX fusion gene, which can activate the Wnt / β-catenin signaling pathway, is one of the causes of synovial sarcoma, and is also the currently recognized gold standard for the diagnosis of synovial sarcoma. At least 95% of synovial sarcomas have t(X; 18)(p11.2; q11.2) chromosomal translocations, of which SS18-SSX1 fusion accounts for the majority (65%), SS18-SSX2 accounts for 35%, and SS18-SSX4 occurs very rarely, all of which are single case reports. Different SS18-SSX fusion genes may represent different molecular genetic mechanisms, which is particularly important for studying the etiology of synovial sarcoma. In addition, patients with SS18-SSX1 fusion have a higher risk of metastasis compared with patients with SS18-SSX2 fusion. Therefore, detecting the type of SS18 fusion gene has important prognostic significance.
[0003] The detection method of SS18-SSX fusion gene in synovial sarcoma can use different levels of technology. Cytogenetic methods can observe t(X;18) translocation, fluorescence in situ hybridization technology can more accurately detect fusion genes, RT-PCR can detect fusion transcripts after translocation, immunohistochemistry can detect heterologous fusion proteins, and NGS can perform whole genome sequencing. These technologies are specific and have diagnostic value. However, traditional cytogenetics requires fresh tumor tissue, and the efficiency of culturing tumor cells is low; PCR technology cannot detect unreported fusion points; NGS testing is expensive; and immunohistochemistry has strong personal subjectivity. At this stage, FISH is a common method for diagnosing synovial sarcoma, but the mainstream product on the market is SS18 break probe, which cannot distinguish fusion types. When the SS18 break FISH test is positive, RT-PCR or second-generation sequencing methods are used to distinguish fusion types or three sets of conventional SS18-SSX dual-color fusion FISH probes are designed to check one by one. SSX1 and SSX4 are only 120kb apart, and these two fusion types are difficult to distinguish with dual-color dual-fusion probes.
[0004] Therefore, there is an urgent need for a convenient and fast detection method that can distinguish different SS18-SSX fusion gene types in one step. Summary of the invention
[0005] In order to solve the above technical problems, the present application provides a fluorescent in situ hybridization probe composition for detecting the SS18-SSX fusion gene of synovial sarcoma, as well as a preparation method and application of the probe composition. When using the probe composition of the present application for diagnosing synovial sarcoma, there is no need to redesign a fusion probe based on the SS18 break for a second step of verification, and different SS18-SSX fusion genes can be distinguished in one step according to the signal type.
[0006] In a first aspect, the present application provides a probe composition for detecting the SS18-SSX fusion gene in synovial sarcoma, the probe composition comprising a SS18 gene 5' probe group, a SS18 gene 3' probe group, a SSX1 gene 3' probe group, and a SSX4 gene 3' probe group. The SS18 gene 5' probe group covers the gene region 380-420 kb upstream of the SS18 gene 5'; the SS18 gene 3' probe group covers the gene region 880-920 kb downstream of the SS18 gene 3'; the SSX1 gene 3' probe group covers the gene region 100-140 kb downstream of the SSX1 gene 3'; the SSX4 gene 3' probe covers the gene region 580-620 kb downstream of the SSX4 gene 3'; and the SS18 gene 5' probe group, SS18 gene 3' probe group, SSX1 gene 3' probe group and SSX4 gene 3' probe group are respectively labeled with four different colors of fluorescence.
[0007] By adopting the technical solution of the present application, the four-color probes designed at 18q11 and Xp11 can distinguish SS18-SSX1 fusion, SS18-SSX2 fusion or SS18-SSX4 fusion according to different signal types. However, the mainstream SS18 break probe on the market cannot distinguish the fusion type. If three sets of two-color fusion probes are designed, it will be time-consuming and labor-intensive, and SSX1 and SSX4 are only 120kb apart, and these two fusion types are difficult to distinguish with two-color fusion probes. The present application can detect the three fusion types of SS18 in one step at the FISH technical level, without the need to design a second-step probe.
[0008] Furthermore, the 5' probe group coverage area of the SS18 gene is: the gene region of approximately 400kb 5' upstream of the SS18 gene, the 3' probe group coverage area of the SS18 gene is: the gene region of approximately 800kb 3' downstream of the SS18 gene, the 3' probe group coverage area of the SSX1 gene is: the gene region of approximately 120kb 3' downstream of the SSX1 gene, and the 3' probe group coverage area of the SSX4 gene is: the gene region of approximately 600kb 3' downstream of the SSX4 gene.
[0009] Furthermore, the SS18 gene 5' probe group covers the region: chr18:23693083-24093124; the SS18 gene 3' probe group covers the region: chr18:22701217-23601423; the SSX1 gene 3' probe group covers the region: chrX:48126879-48246957; the SSX4 gene 3' probe group covers the region: chrX:48287785-48887401.
[0010] Furthermore, the probes in the probe set are 40-60 bp in length.
[0011] Furthermore, the SS18 gene 5' probe group is labeled with a red fluorescent group, the SS18 gene 3' probe group is labeled with a green fluorescent group, the SSX1 gene 3' probe group is labeled with a yellow fluorescent group, and the SSX4 gene 3' probe group is labeled with a cyan fluorescent group.
[0012] Using the above technical solution, in normal female cells without SS18-SSX fusion, two red-green overlapping signals and two yellow-cyan overlapping signals are expected. In normal male cells without SS18-SSX fusion, two red-green overlapping signals and one yellow-cyan overlapping signal are expected. When red and green are separated, it indicates that the SS18 locus is broken. If a red signal co-localizes with a yellow-cyan signal and a single green signal appears, it indicates SS18-SSX1 fusion. If a green signal co-localizes with or is adjacent to a yellow-cyan signal and a single red signal appears, it indicates SS18-SSX2 fusion. If a red signal co-localizes with a cyan signal and a green signal co-localizes with a yellow signal, it indicates SS18-SSX4 fusion.
[0013] In a second aspect, the present application provides a method for preparing the probe composition according to the first aspect, the preparation method comprising the following steps: Step 1, respectively obtaining the genomic non-repetitive sequences of the regions covered by the SS18 gene 5' probe group, the SS18 gene 3' probe group, the SSX1 gene 3' probe group, and the SSX4 gene 3' probe group; Step 2, dividing the non-repetitive genomic sequences into 1 kb blocks respectively, and performing probe design and screening by OligoArray software to obtain SS18 gene 5' probe pool, SS18 gene 3' probe pool, SSX1 gene 3' probe pool and SSX4 gene 3' probe pool; Step 3, respectively adding a label sequence to the 5' end and the 3' end of each probe sequence in the probe pool to obtain a SS18 gene 5' probe pool, a SS18 gene 3' probe pool, a SSX1 gene 3' probe pool and a SSX4 gene 3' probe pool containing a label sequence; Step 4, respectively chemically synthesizing the probes in the SS18 gene 5' probe pool, the SS18 gene 3' probe pool, the SSX1 gene 3' probe pool and the SSX4 gene 3' probe pool, and mixing them evenly to obtain a SS18 gene 5' probe library, a SS18 gene 3' probe library, a SSX1 gene 3' probe library and a SSX4 gene 3' probe library; Step 5, using universal primers containing different fluorescent groups at the 5' end to perform amplification and labeling reaction on the middle probes of the SS18 gene 5' probe library, SS18 gene 3' probe library, SSX1 gene 3' probe library and SSX4 gene 3' probe library, to obtain the SS18 gene 5' probe group, SS18 gene 3' probe group, SSX1 gene 3' probe group and SSX4 gene 3' probe group.
[0014] Furthermore, the screening in step 2 is as follows: the probe length is 40-60 bp, the TM value is 85-99°C, the GC ratio is 40-80%, excluding TTTT / GGGG / AAAA / CCCC, and the minimum probe interval is 5 bp.
[0015] Furthermore, the tag sequence in step 3 is: the 17 bp tag sequence at the 5' end is: 5'-TGTAAAACGACGGCCAG-3' (SEQ ID NO. 1), and the 18 bp tag sequence at the 3' end is: 5'-GGTCATAGCTGTTTCCTG-3' (SEQ ID NO. 2).
[0016] Furthermore, the chemical synthesis in step 4 is performed using a DNA synthesizer.
[0017] Furthermore, the universal primers in step 5 are 5'-TGTAAAACGACGGCCAGT-3' (SEQ ID NO. 3) and 5'-CAGGAAACAGCTATGACC-3' (SEQ ID NO. 4).
[0018] In a third aspect, the present application also provides a kit for detecting the SS18-SSX fusion gene of synovial sarcoma, comprising the probe composition described in the first aspect of the present application and a hybridization buffer.
[0019] Furthermore, the hybridization buffer comprises 10 wt% deionized formamide, 20 wt% dextran sulfate, 1 μg / mL RNaseA, 0.6 M sodium chloride, and 10 mM citric acid buffer.
[0020] In a fourth aspect, the present application also provides the use of the probe composition described in the first aspect or the kit described in the third aspect in detecting different fusion gene types of SS18-SSX in synovial sarcoma.
[0021] Furthermore, the application is specifically to mix the probe set with a hybridization buffer, and then co-denature and hybridize with the sample to be detected, wash and then examine under a microscope, and when red and green are separated, it indicates that the SS18 locus is broken, and if a red signal co-localizes with a yellow-cyan signal and a single green signal appears, it indicates SS18-SSX1 fusion. If a green signal co-localizes with or is adjacent to a yellow-cyan signal and a single red signal appears, it indicates SS18-SSX2 fusion. If a red signal co-localizes with a cyan signal and a green signal co-localizes with a yellow signal, it indicates SS18-SSX4 fusion.
[0022] In summary, the present application has the following technical effects compared with the prior art.
[0023] 1) In the probe composition of the present application, the SS18 probe composition contains two regions marked with different colors. When the two color signals of the SS18 gene are separated, it indicates that the SS18 gene is fused with other genes. At the same time, since SSX1 and SSX4 are close to each other, it is difficult to distinguish them using conventional two-color fusion probes. The probe composition of the present application carries four different fluorescent markers, which can distinguish between the rare SS18-SSX4 fusion and the common SS18-SSX1 fusion.
[0024] 2) When the probe composition of the present application is used for the diagnosis of synovial sarcoma, different SS18-SSX fusion genes can be distinguished in one step according to the signal type, without the need to design a fusion probe for a second step of verification based on the SS18 break. The pathogenesis can also be explained and the prognosis can be judged according to different fusion types.
[0025] 3) Compared with traditional BAC clone FISH, the probe set preparation method of the present application is completely independent of BAC clones, contains no repetitive sequences, and does not require expensive Human cot1 DNA for blocking, which greatly reduces the hybridization background and preparation costs.
[0026] 4) Compared with the preparation method of rapid fluorescent in situ hybridization probes without repetitive sequences established in recent years, the screening of probes in the present application mainly relies on the program, and a more stable PCR method is used to amplify and label the probes. The probe labeling rate is higher and more uniform, and fluorescent labeling is only performed on the 5' end of the probe, so it does not affect the hybridization pairing reaction of the probe; in addition, the present invention does not use nick translation reaction and random primer reaction that are easily affected by the environment or operating techniques to label the probe, which increases the stability between batches, and the probe in situ hybridization time is short, and the signal-to-noise ratio and specificity are high. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] For better understanding and implementation, the present invention is described in detail below with reference to the accompanying drawings.
[0028] Figure 1 This is a schematic diagram of the positioning pattern of the SS18-SSX fusion probe of the present invention.
[0029] Figure 2 It is a schematic diagram of the application of cell-negative cases in the experimental examples of the present invention.
[0030] Figure 3 This is a schematic diagram of the SS18-SSX1 fusion case in the experimental example of the present invention.
[0031] Figure 4 This is a schematic diagram of the SS18-SSX2 fusion case in the experimental example of the present invention.
[0032] Figure 5This is a theoretical schematic diagram of the SS18-SSX4 fusion case of the present invention. DETAILED DESCRIPTION
[0033] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, conditions described in laboratory manuals or conditions recommended by manufacturers.
[0034] Example 1 Preparation of SS18 gene 5' probe set The method for preparing the SS18 gene 5' probe set comprises the following steps: Step 1. In the chromosome, the SS18 gene region refers to chr18:23,596,217-23,670,611. Download the genomic non-repetitive sequence of about 400kb (chr18:23693083-24093124) upstream of the SS18 gene 5' from the UCSC Genome Browser, which is the coverage area of the SS18 gene 5' probe group.
[0035] Step 2: Use the perl plug-in program chunks.pl to divide the non-repetitive genomic sequence in the area covered by the SS18 gene 5' probe obtained in step (1) into blocks of 1 kb in size; import the divided blocks into OligoArray software in batches for probe design and probe screening. The conditions for probe screening are: probe length 50 bp, TM value 85-99°C, GC ratio 40-80%, no TTTT / GGGG / AAAA / CCCC, and the minimum probe interval is 5 bp. Then export the screened probes to an EXCEL table to obtain a SS18 gene 5' probe pool.
[0036] Step 3, add a 17 bp tag sequence to the 5' end and a 18 bp tag sequence to the 3' end of each probe, the 17 bp tag sequence at the 5' end is: 5'-TGTAAAACGACGGCCAG-3' (SEQ ID NO.1); the 18 bp tag sequence at the 3' end is: 5'-GGTCATAGCTGTTTCCTG-3' (SEQ ID NO.2), and a series of SS18 gene 5' probe sequence pools with tag sequences are obtained.
[0037] Step 4: Use a DNA synthesizer to chemically synthesize a series of SS18 gene 5' probe sequences obtained in step 3, mix the chemically synthesized probes, and prepare a SS18 gene 5' probe library with sequence tags.
[0038] Step 5, synthesize a universal primer pair with a red fluorescent group at the 5' end, the sequence of the universal primer pair is 5'-TGTAAAACGACGGCCAGT-3' (SEQ ID NO.3) and 5'-CAGGAAACAGCTATGACC-3' (SEQ ID NO.4), and use the universal primer with a red fluorescent group at the 5' end to perform an amplification and labeling reaction on the SS18 gene 5' probe library. The amplification and labeling reaction system is shown in Table 1. The conditions of the amplification and labeling reaction are: 95°C for 5min; 94°C for 30s, 58°C for 30s, 72°C for 30s, for a total of 35 cycles; 72°C for 10min.
[0039] Table 1 Amplification and labeling reaction PCR system Reagents volume 2×PCR Mix 25μL template 50ng Universal primers 2μL <![CDATA[H2O]]> Make up to 50 μL The amplified labeled product was purified and diluted to obtain a fluorescently labeled SS18 gene 5' probe set, with a probe set concentration of 2-5 ng / μL.
[0040] Example 2 Preparation of SS18 gene 3' probe set The method for preparing the SS18 gene 3' probe set comprises the following steps: Step 1. In the chromosome, the SS18 gene region refers to chr18:23,596,217-23,670,611. Download the genomic non-repetitive sequence of about 900kb (chr18:22,701,217-23,601,423) downstream of the 3' of the SS18 gene from the UCSC Genome Browser, which is the coverage area of the SS18 gene 3' probe group.
[0041] Steps 2-4 are the same as in Example 1.
[0042] Step 5: synthesize a universal primer pair with a green fluorescent group at the 5' end, and the remaining steps are the same as step 5 to obtain a fluorescently labeled SS18 gene 3' probe set with a probe set concentration of 2-5 ng / μL.
[0043] Example 3 Preparation of SSX1 gene 3' probe set The method for preparing the SSX1 gene 3' probe set comprises the following steps: Step 1. In the chromosome, the SSX1 gene region refers to chrX:48,114,797-48,126,879. The genomic non-repetitive sequence of about 120 kb (chrX:48,126,879-48,246,957) downstream of the 3' of the SSX1 gene was downloaded from the UCSC Genome Browser, which is the coverage area of the 3' probe group of the SSX1 gene.
[0044] Steps 2-4 are the same as in Example 1.
[0045] Step 5: synthesize a universal primer pair with a yellow fluorescent group at the 5' end, and the remaining steps are the same as step 5 to obtain a fluorescently labeled SSX1 gene 3' probe set with a probe set concentration of 2-5 ng / μL.
[0046] Example 4 Preparation of SSX4 gene 3' probe set The method for preparing the SSX4 gene 3' probe set comprises the following steps: Step 1. In the chromosome, the SS18 gene region refers to the SSX4 gene region refers to chrX:48,242,957-48,252,785. Download the genomic non-repetitive sequence of about 600kb (chrX:48,287,785-48,887,401) downstream of the 3' of the SSX1 gene from the UCSC Genome Browser, which is the coverage area of the 3' probe set of the SSX1 gene.
[0047] Steps 2-4 are the same as in Example 1.
[0048] Step 5: synthesize a universal primer pair with a cyan fluorescent group at the 5' end, and the remaining steps are the same as step 5 to obtain a fluorescently labeled SSX4 gene 3' probe set with a probe set concentration of 2-5 ng / μL.
[0049] Example 5 Probe Composition and Kit The fluorescently labeled SS18 gene 5' probe group, SS18 gene 3' probe group, SSX1 gene 3' probe group, and SSX4 gene 3' probe group were mixed in equal volume ratios to obtain a probe composition for detecting the SS18-SSX fusion gene of synovial sarcoma. The probe composition was mixed with a hybridization buffer composition to form a kit for detecting different types of SS18-SSX fusion genes of synovial sarcoma.
[0050] The components of the hybridization buffer are: 10 wt% deionized formamide, 20 wt% dextran sulfate, 1 μg / mL RNase A, 0.6 M sodium chloride, and 10 mM citric acid buffer.
[0051] Example 6 Detection of samples to be tested (1) Sample processing: The “Sample Releaser” kit (Cat. No.: CL-003, Registration No.: E-Han Xie Bei 20160084) of Wuhan Kanglu Biotechnology Co., Ltd. was used for pre-processing, and cell-negative cases were used as controls.
[0052] (2) Preparation of SS18-SSX fusion gene probe hybridization mixture: The fluorescently labeled SS18 gene 5' probe group, SS18 gene 3' probe group, SSX1 gene 3' probe group, SSX4 gene 3' probe group prepared in Examples 1-4 and hybridization buffer were mixed in a volume ratio of 1:1:1:1:7, wherein the components of the hybridization buffer were: 10 wt% deionized formamide, 20 wt% dextran sulfate, 1 μg / mL RNaseA, 0.6 M sodium chloride, and 10 mM citric acid buffer.
[0053] (3) Probe sample co-denaturation: Each sample in step (1) was hybridized with 10 μL of the SS18-SSX fusion gene probe hybridization mixture prepared in step (2), and the sample was covered with a 22×22 mm cover glass and sealed with rubber glue. After sealing, the cover glass was placed in a hybridizer for denaturation at 85°C for 5 min, and hybridization was performed at 42°C for 45 min.
[0054] (4) Post-hybridization washing: After hybridization is completed, remove the coverslip and place the slide in a 60°C preheated washing solution to wash away unbound probes.
[0055] (5) Restaining and microscopic examination: Add 10 μL of antifade mounting medium to the dried slides, and then observe the hybridization results under a fluorescence microscope.
[0056] The results are as follows Figure 2-5 As shown, Figure 2 As shown, the red and green signals in the cell-negative cases were not separated, indicating that the SS18 locus was not broken; Figure 3 As shown in the figure, the separation of red and green indicates that the SS18 locus is broken, and the co-localization of a red signal and a yellow-cyan signal and a single green signal indicate SS18-SSX1 fusion; Figure 4 As shown in the figure, the red and green separation indicates that the SS18 locus is broken, and the co-localization or proximity of a green signal and a yellow-cyan signal, as well as a single red signal, indicates SS18-SSX2 fusion. Since there are very few cases of SS18-SSX4 fusion, this gene fusion phenomenon was not detected in the test samples. Figure 5 This is a theoretical schematic diagram of the SS18-SSX4 fusion case of the present invention. If a red signal co-localizes with a cyan signal, and a green signal co-localizes with a yellow signal, it indicates SS18-SSX4 fusion.
[0057] In summary, when the probe composition of the present application is used for the diagnosis of synovial sarcoma, different fusion genes of SS18-SSX can be distinguished in one step according to the signal type, without the need to design a fusion probe based on the SS18 break for a second step of verification. The probe composition and detection method of the present application provide a new diagnostic and prognostic approach for the clinical diagnosis of synovial sarcoma, and have a high application value.
[0058] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.
Claims
1. A probe composition for detecting SS18-SSX fusion gene of synovial sarcoma, characterized in that: The probe composition includes a SS18 gene 5' probe group, a SS18 gene 3' probe group, a SSX1 gene 3' probe group and a SSX4 gene 3' probe group, The SS18 gene 5' probe group covers the gene region of 380-420 kb 5' upstream of the SS18 gene; the SS18 gene 3' probe group covers the gene region of 880-920 kb 3' downstream of the SS18 gene; the SSX1 gene 3' probe group covers the gene region of 100-140 kb 3' downstream of the SSX1 gene; the SSX4 gene 3' probe covers the gene region of 580-620 kb 3' downstream of the SSX4 gene; The SS18 gene 5' probe set, the SS18 gene 3' probe set, the SSX1 gene 3' probe set and the SSX4 gene 3' probe set are respectively labeled with four different colors of fluorescence.
2. The probe composition according to claim 1, characterized in that The SS18 gene 5' probe group covers the gene region of approximately 400 kb 5' upstream of the SS18 gene, the SS18 gene 3' probe group covers the gene region of approximately 800 kb 3' downstream of the SS18 gene, the SSX1 gene 3' probe group covers the gene region of approximately 120 kb 3' downstream of the SSX1 gene, and the SSX4 gene 3' probe group covers the gene region of approximately 600 kb 3' downstream of the SSX4 gene.
3. The probe composition according to claim 2, characterized in that The SS18 gene 5' probe set covers the region: chr18:23693083-24093124; The SS18 gene 3' probe set covers the region: chr18:22701217-23601423; The SSX1 gene 3' probe set covers the region: chrX:48126879-48246957; The SSX4 gene 3' probe set covers the region: chrX:48287785-48887401.
4. The probe composition according to claim 1, characterized in that The probes in the probe set are 40-60 bp in length.
5. The probe composition according to claim 1, characterized in that The SS18 gene 5' probe group is labeled with a red fluorescent group, the SS18 gene 3' probe group is labeled with a green fluorescent group, the SSX1 gene 3' probe group is labeled with a yellow fluorescent group, and the SSX4 gene 3' probe group is labeled with a cyan fluorescent group.
6. The method for preparing the probe composition according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: Step 1, respectively obtaining the genomic non-repetitive sequences of the regions covered by the SS18 gene 5' probe group, the SS18 gene 3' probe group, the SSX1 gene 3' probe group, and the SSX4 gene 3' probe group; Step 2, dividing the non-repetitive genomic sequences into 1 kb blocks respectively, and performing probe design and screening by using OligoArray software to obtain SS18 gene 5' probe pool, SS18 gene 3' probe pool, SSX1 gene 3' probe pool and SSX4 gene 3' probe pool; Step 3, adding a tag sequence to the 5' end and the 3' end of each probe sequence in the probe pool, respectively, to obtain a SS18 gene 5' probe pool, a SS18 gene 3' probe pool, a SSX1 gene 3' probe pool and a SSX4 gene 3' probe pool containing a tag sequence; Step 4, chemically synthesizing the probes in the SS18 gene 5' probe pool, SS18 gene 3' probe pool, SSX1 gene 3' probe pool and SSX4 gene 3' probe pool respectively, and mixing them evenly to obtain the SS18 gene 5' probe library, SS18 gene 3' probe library, SSX1 gene 3' probe library and SSX4 gene 3' probe library; Step 5, using universal primers containing different fluorescent groups to perform amplification and labeling reaction on the middle probes of the SS18 gene 5' probe library, SS18 gene 3' probe library, SSX1 gene 3' probe library and SSX4 gene 3' probe library, to obtain the SS18 gene 5' probe group, SS18 gene 3' probe group, SSX1 gene 3' probe group and SSX4 gene 3' probe group.
7. The preparation method according to claim 6, characterized in that: The screening in step 2 is as follows: the probe length is 40-60 bp, the TM value is 85-99°C, the GC ratio is 40-80%, excluding TTTT / GGGG / AAAA / CCCC, and the minimum probe interval is 5 bp.
8. The preparation method according to claim 6, characterized in that: The tag sequence in step 3 is: the 17 bp tag sequence at the 5' end is: 5'-TGTAAAACGACGGCCAG-3' (SEQ ID NO.1), and the 18 bp tag sequence at the 3' end is: 5'-GGTCATAGCTGTTTCCTG-3' (SEQ ID NO.2); the universal primers in step 5 are 5'-TGTAAAACGACGGCCAGT-3' (SEQ ID NO.3) and 5'-CAGGAAACAGCTATGACC-3' (SEQ ID NO.4).
9. A kit for detecting SS18-SSX fusion gene in synovial sarcoma, characterized in that: The kit contains the probe composition according to any one of claims 1 to 5 and a hybridization buffer.
10. Use of the probe composition according to any one of claims 1 to 5 or the kit according to claim 9 in detecting different fusion gene types of SS18-SSX in synovial sarcoma.