Probe composition for detecting different fusion genes of NUP214 as well as preparation method and application of probe composition

Abstract

The invention belongs to the field of molecular biology, and relates to a probe composition for detecting different fusion genes of NUP214 as well as a preparation method and application of the probe composition. The invention provides a probe composition for detecting different fusion genes of NUP214, the probe composition comprises an NUP214 gene 3'probe group, an ABL1 gene 3 'probe group and an SET gene 5' probe group, and the NUP214 gene 3 'probe group, the ABL1 gene 3' probe group and the SET gene 5 'probe group are respectively marked by adopting three different colors of fluorescence. By adopting the technical scheme provided by the invention, three sections of 9q34 are designed into probes with different fluorescence, and DEK-NUP214 fusion or NUP214-ABL1 fusion or SET-NUP214 fusion can be distinguished according to different signal types; the DEK-NUP214 fusion can only be distinguished by using a traditional fusion probe, and the NUP214-ABL1 fusion and the SET-NUP214 fusion cannot be distinguished by using the traditional fusion probe. Different fusion genes of NUP214 are different in leukemia diagnosis and clinical treatment, so that the distinguishing of different fusion genes has clinical guiding significance.

Description

Technical Field

[0001] The present application belongs to the field of molecular biology, and specifically relates to a probe composition for detecting different fusion genes of NUP214, and a preparation method and application thereof. Background Art

[0002] Detection of different fusion genes of NUP214 is one of the methods to diagnose different types of leukemia, clarify the pathogenesis, and guide clinical treatment and medication. Chromosomal translocations involving the NUP214 locus frequently occur in acute leukemia, and there are three common partner genes-DEK, ABL1, and SET, and each has three different fusion forms. Among them, t(6;9)(p22;q34) balanced translocation leads to DEK-NUP214 fusion; 9q34 free loop amplification leads to NUP214-ABL1 fusion (that is, after ABL1 and NUP214 break, the genome segment is circularized, generating NUP214-ABL1 fusion gene, circularized fragments, and constantly self-replicate to form many copies); del(9)(q34.11q34.13) deletion leads to SET-NUP214 fusion, and the latter two fusion forms cannot be detected by conventional cytogenetic methods and are occult fusions.

[0003] NUP214-ABL1 fusion and SET-NUP214 fusion are mainly associated with acute T-lymphocytic leukemia (T-ALL), while DEK-NUP214 fusion is mainly associated with acute myeloid leukemia (AML), indicating different leukemic driving mechanisms. WHO has added AML with t(6;9)(p22;q34):DEK-NUP214 to the classification system for tumors of hematopoietic and lymphoid tissues as an independent clinical subtype of AML. This type of disease has a very poor prognosis and a high risk of relapse. Bone marrow transplantation should be performed as soon as possible after early remission. The NCCN guideline risk category also classifies this as a poor / unfavorable group. Patients carrying the NUP214-ABL1 fusion gene usually have high-risk factors for T-ALL, including elevated white blood cell counts, mediastinal masses, and extramedullary involvement. They usually relapse early and have a poor prognosis. In addition, its fusion protein has tyrosine kinase activity and can benefit from tyrosine kinase inhibitor treatment. The SET-NUP214 fusion gene inhibits T cell maturation through transcriptional activation of the NUP214 gene, thereby promoting the pathogenesis of T-ALL. Therefore, distinguishing different NUP214 fusion genes has clinical significance.

[0004] There are currently four methods for detecting fusion genes: whole genome sequencing (WGS), transcriptome sequencing (RNA-seq), real-time quantitative PCR (qRT-PCR), and fluorescence in situ hybridization (FISH). The first two methods are difficult to promote in most laboratories due to their high cost and high sample requirements. The latter two methods are currently the commonly used methods for detecting fusion genes. However, NUP214 fusion genes have many forms, including DEK-NUP214 fusion caused by t(6;9)(p22;q34) balanced translocation, NUP214-ABL1 fusion caused by 9q34 episome amplification free ring amplification, and SET-NUP214 fusion caused by del(9)(q34.11q34.13). PCR technology is relatively difficult to detect and cannot detect unreported fusion points. FISH technology can very intuitively reflect the fusion status of the two genes for the detection of fusion genes. It can detect different fusion points that have been reported and unreported, overcoming the shortcomings of PCR technology. However, the three fusion genes NUP214 have three different fusion forms, which require the design of several sets of conventional fusion probes to detect; and SET, ABL1 and NUP214 are all located at 9q34, and the chromosome positions are close to each other, so it is difficult to detect the fusion gene with conventional FISH probes.

[0005] Therefore, there is an urgent need for a detection method that can quickly and easily detect the three different fusion genes of NUP214. Summary of the invention

[0006] In order to solve the above technical problems, the present application provides a fluorescent in situ hybridization probe composition for detecting three different fusion genes of NUP214, as well as a preparation method and application of the probe composition.

[0007] In a first aspect, the present application provides a probe composition for detecting different fusion genes of NUP214, wherein the probe composition comprises a NUP214 gene 3' probe group, an ABL1 gene 3' probe group, and a SET gene 5' probe group.

[0008] The NUP214 gene 3' probe group covers the gene region of 500-540 kb 3' downstream of the NUP214 gene, the ABL1 gene 3' probe group covers the gene region of 230-240 kb 3' downstream of the ABL1 gene, and the SET gene 5' probe group covers the gene region of 490-510 kb 5' upstream of the SET gene; and the NUP214 gene 3' probe group, the ABL1 gene 3' probe group and the SET gene 5' probe group are respectively labeled with three different colors of fluorescence.

[0009] By adopting the technical solution of the present application, by designing the three segments of 9q34 as probes with different fluorescence, when diagnosing different types of leukemia, it is possible to distinguish whether it is DEK-NUP214 fusion, NUP214-ABL1 fusion or SET-NUP214 fusion according to the type of signal when breakage and fusion occur.

[0010] Furthermore, the NUP214 gene 3' probe group covers the gene region of about 520 kb 3' downstream of the NUP214 gene, the ABL1 gene 3' probe group covers the gene region of about 238 kb 3' downstream of the ABL1 gene, and the SET gene 5' probe group covers the gene region of about 500 kb 5' upstream of the SET gene.

[0011] Furthermore, the NUP214 gene 3' probe group covers the region: chr9: 134156687-134676106, the ABL1 gene 3' probe group covers the region: chr9: 133763062-134001268, and the SET gene 5' probe group covers the region: chr9: 130945934-131445934.

[0012] Furthermore, the probes in the probe set are 40-60 bp in length.

[0013] Furthermore, the probe composition includes a NUP214 gene 3' probe group labeled with a green fluorescent group, an ABL1 gene 3' probe group labeled with a red fluorescent group, and a SET gene 5' probe group labeled with a cyan fluorescent group.

[0014] Using the above technical solution, when there is no abnormality in 9q34, the signal pattern is a signal pattern of red and green overlapping and adjacent to cyan. When red and green are separated, it indicates that the NUP214 locus is broken. If the red dot is adjacent to the cyan dot after separation, it indicates DEK-NUP214 fusion (that is, after the NUP214 at 9q34 is broken, the 3' end green dot is translocated to the 6p22 DEK gene, leaving the original red and green signal dot at 9q34). If the green dot is adjacent to the cyan dot after separation, and the red dot is amplified, it indicates NUP214-ABL1 fusion (that is, after the NUP214 and ABL1 are broken, the 5' end of the NUP214 gene is used as the head and the 3' end of the ABL1 gene is used as the tail, and circularization is performed to form a free ring carrying the NUP214-ABL1 fusion, and the free ring is amplified again). If the red dot is missing and the green dot is fused with the cyan dot, it indicates SET-NUP214 fusion.

[0015] In a second aspect, the present application also 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 NUP214 gene 3' probe group, the ABL1 gene 3' probe group, and the SET gene 5' 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 a NUP214 gene 3' probe pool, an ABL1 gene 3' probe pool, and a SET gene 5' 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 NUP214 gene 3' probe pool, an ABL1 gene 3' probe pool, and a SET gene 5' probe pool containing tag sequences; Step 4, chemically synthesizing the probes in the NUP214 gene 3' probe pool, the ABL1 gene 3' probe pool and the SET gene 5' probe pool respectively, and mixing them evenly to obtain the NUP214 gene 3' probe library, the ABL1 gene 3' probe library and the SET gene 5' probe library; Step 5: Use universal primers containing different fluorescent groups to perform amplification and labeling reaction on the probes in the NUP214 gene 3' probe library, the ABL1 gene 3' probe library and the SET gene 5' probe library to obtain the NUP214 gene 3' probe group, the ABL1 gene 3' probe group and the SET gene 5' probe group.

[0016] 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.

[0017] 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).

[0018] Furthermore, the chemical synthesis in step 4 is performed using a DNA synthesizer.

[0019] Furthermore, the universal primers in step 5 are 5'-TGTAAAACGACGGCCAGT-3' (SEQ ID NO. 3) and 5'-CAGGAAACAGCTATGACC-3' (SEQ ID NO. 4).

[0020] In a third aspect, the present application also provides a kit for detecting different fusion genes of NUP214, wherein the kit contains the probe composition and hybridization buffer described in the first aspect.

[0021] 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.

[0022] 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 genes of NUP214.

[0023] Furthermore, the application is specifically to mix the probe set with hybridization buffer, and then co-denature and hybridize with the sample to be detected, wash and then examine under a microscope. When there is no abnormality in 9q34, the signal pattern is a signal pattern of red and green overlapping and adjacent to cyan. When red and green are separated, it indicates that the NUP214 locus is broken. If the red dot is adjacent to the cyan dot after separation, it indicates DEK-NUP214 fusion (that is, after the NUP214 at 9q34 is broken, the 3' end green dot is translocated to the 6p22 DEK gene, leaving the original red and cyan signal dot at 9q34). If the green dot is adjacent to the cyan dot after separation, and the red dot is amplified, it indicates NUP214-ABL1 fusion (that is, after the NUP214 and ABL1 are broken, the 5' end of the NUP214 gene is used as the head and the 3' end of the ABL1 gene is used as the tail, and circularization is performed to form a free ring carrying the NUP214-ABL1 fusion, and the free ring is amplified again). If the red dot is missing and the green dot is merged with the cyan dot, it indicates SET-NUP214 fusion.

[0024] In summary, the present application has the following technical effects compared with the prior art.

[0025] 1) The probe primer set of the present application, by designing the three segments of 9q34 as probes with different fluorescence, can distinguish DEK-NUP214 fusion, NUP214-ABL1 fusion or SET-NUP214 fusion according to the type of signal when a breakage fusion occurs during the diagnosis of different types of leukemia, wherein the two fusion forms of NUP214-ABL1 fusion and SET-NUP214 fusion cannot be detected by conventional cytogenetics.

[0026] 2) 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.

[0027] 3) 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

[0028] For better understanding and implementation, the present invention is described in detail below with reference to the accompanying drawings.

[0029] Figure 1 This is a schematic diagram of the three-color probe positioning mode of the present invention.

[0030] Figure 2 It is a schematic diagram of the application of cell-negative cases in the experimental examples of the present invention.

[0031] Figure 3 This is a schematic diagram of the DEK-NUP214 fusion case in the experimental example of the present invention.

[0032] Figure 4 This is a schematic diagram of the NUP214-ABL1 fusion case in the experimental example of the present invention.

[0033] Figure 5 This is a schematic diagram of the SET-NUP214 fusion case in the experimental example of the present invention. DETAILED DESCRIPTION

[0034] 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.

[0035] Example 1 Preparation of NUP214 gene 3' probe set The method for preparing the NUP214 gene 3' probe set comprises the following steps: Step 1. In the chromosome, the NUP214 gene region refers to chr9:134000981-134109091. Download the genomic non-repetitive sequence of about 520 kb (chr9:134156687-134676106) downstream of the 3' of the NUP214 gene from the UCSC Genome Browser, which is the coverage area of ​​the 3' probe set of the NUP214 gene.

[0036] Step 2: Use the perl plug-in program chunks.pl to divide the non-repetitive genomic sequences in the region covered by the NUP214 gene 3' probe set obtained in step (1) into 1 kb blocks; 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 a minimum probe interval of 5 bp. Then export the screened probes to an EXCEL table to obtain a NUP214 gene 3' probe pool.

[0037] 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 NUP214 gene 3' probe sequence pools with tag sequences are obtained.

[0038] Step 4: Use a DNA synthesizer to chemically synthesize a series of NUP214 gene 3' probe sequences obtained in step 3, mix the chemically synthesized probes, and prepare a NUP214 gene 3' probe library with sequence tags.

[0039] Step 5, synthesize a universal primer pair with a green 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 green fluorescent group at the 5' end to perform amplification and labeling reaction on the 3' probe library of the NUP214 gene. 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.

[0040] Table 1 Amplification and labeling reaction PCR system Reagents volume 2×PCR Mix 25μL template 50ng Universal primers 2μL <![CDATA[H 2 The]]> Make up to 50 μL The amplified labeled product was purified and diluted to obtain a NUP214 gene 3' probe set with a green fluorescent label, and the concentration of the probe set was 2-5 ng / μL.

[0041] Example 2 Preparation of ABL1 gene 3' probe set The method for preparing the ABL1 gene 3' probe set comprises the following steps: Step 1. In the chromosome, the ABL1 gene region refers to chr9:133589268-133763062. Download the genomic non-repetitive sequence of about 238 kb (chr9:133763062-134001268) downstream of the 3' of the ABL1 gene from UCSC GenomeBrowser, which is the coverage area of ​​the 3' probe set of the ABL1 gene.

[0042] Steps 2-4 are the same as in Example 1.

[0043] Step 5, synthesize a universal primer pair with a red fluorescent group at the 5' end, and the remaining steps are the same as step 5 to obtain a 3' probe set of the ABL1 gene with a red fluorescent label, and the concentration of the probe set is 2-5 ng / μL.

[0044] Example 3 Preparation of SET gene 5' probe set The method for preparing the SET gene 5' probe set comprises the following steps: Step 1: In the chromosome, the SET gene 5' region refers to chr9:131445934-131458675. The genomic non-repetitive sequence of about 500 kb (chr9:130945934-131445934) upstream of the SET gene 5' is downloaded from UCSC Genome Browser, which is the coverage area of ​​the SET gene 5' probe group.

[0045] Steps 2-4 are the same as in Example 1.

[0046] 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 SET gene 5' probe group with a cyan fluorescent label, and the concentration of the probe group is 2-5 ng / μL.

[0047] Example 4 Probe Composition and Kit The fluorescently labeled NUP214 gene 3' probe group, ABL1 gene 3' probe group and SET gene 5' probe group were mixed in equal volumes to obtain a probe composition for detecting different fusion genes of NUP214, and the probe composition was mixed with a hybridization buffer composition to form a kit for detecting different fusion genes of NUP214. The components of the hybridization buffer are: 10wt% deionized formamide, 20wt% dextran sulfate, 1μg / mL RNaseA, 0.6M sodium chloride, and 10mM citric acid buffer.

[0048] Example 5 Detection of samples to be tested (1) Sample processing: Place the blood cell slide sample in a container containing 2×SSC, heat it in a microwave oven at high temperature for 3 minutes until the liquid boils, and then continue to heat it at medium-low temperature for 10 minutes. After the treatment is completed, immediately place the slide in 20°C pre-cooled gradient alcohol to dehydrate and dry it for later use.

[0049] (2) Preparation of hybridization probe mixtures for detecting different fusion genes of NUP214: The NUP214 gene 3' probe group, ABL1 gene 3' probe group and SET gene 5' probe group prepared in Examples 1-3 were respectively mixed with hybridization buffer at a volume ratio of 1:1:1:8, 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.

[0050] (3) Co-denaturation of probe samples: Each sample in step (1) was hybridized with 10 μL of the NUP214 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 88°C for 2 min, and hybridization was performed at 45°C for 45 min.

[0051] (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.

[0052] (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.

[0053] The results are as follows Figure 2-5 As shown, Figure 2 As shown, in the cell-negative case, when there is no abnormality in 9q34, the signal pattern is a signal pattern of red and green overlapping and adjacent to cyan. Figure 3 As shown in the figure, when the red and green are separated, it indicates that the NUP214 gene locus is broken. If the red dot is adjacent to the blue dot after separation, it indicates that it is a DEK-NUP214 fusion (that is, after the NUP214 at 9q34 is broken, the 3' end green dot is translocated to the 6p22 DEK gene, leaving the original red and blue signal point at 9q34). Figure 4 As shown in the figure, if the green dot is adjacent to the blue dot after separation, and the red dot is amplified, it indicates NUP214-ABL1 fusion (that is, after NUP214 and ABL1 are broken, the 5' end of the NUP214 gene is used as the head and the 3' end of the ABL1 gene is used as the tail, and circularization is performed to form a free ring carrying the NUP214-ABL1 fusion, and the free ring is amplified again). Figure 5 As shown, if the red dot is missing and the green dot is merged with the cyan dot, it indicates SET-NUP214 fusion.

[0054] In summary, when the probe composition of the present application is used to diagnose different types of leukemia, three groups of different fusion genes of NUP214 can be detected and distinguished according to the type of signal, which provides a new diagnostic and prognostic approach for the clinical diagnosis of leukemia and has a high application value.

[0055] 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 different fusion genes of NUP214, characterized in that: The probe composition includes a NUP214 gene 3' probe group, an ABL1 gene 3' probe group and a SET gene 5' probe group, The NUP214 gene 3' probe group covers the gene region of 500-540 kb 3' downstream of the NUP214 gene, the ABL1 gene 3' probe group covers the gene region of 230-240 kb 3' downstream of the ABL1 gene, and the SET gene 5' probe group covers the gene region of 490-510 kb 5' upstream of the SET gene; The NUP214 gene 3' probe group, the ABL1 gene 3' probe group and the SET gene 5' probe group are respectively labeled with three different colors of fluorescence.

2. The probe composition according to claim 1, characterized in that The NUP214 gene 3' probe group covers the gene region of about 520 kb 3' downstream of the NUP214 gene, the ABL1 gene 3' probe group covers the gene region of about 238 kb 3' downstream of the ABL1 gene, and the SET gene 5' probe group covers the gene region of about 500 kb 5' upstream of the SET gene.

3. The probe composition according to claim 2, characterized in that The NUP214 gene 3' probe group covers the region: chr9: 134156687-134676106, the ABL1 gene 3' probe group covers the region: chr9: 133763062-134001268, and the SET gene 5' probe group covers the region: chr9: 130945934-131445934.

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 probe composition comprises a NUP214 gene 3' probe group labeled with a green fluorescent group, an ABL1 gene 3' probe group labeled with a red fluorescent group, and a SET gene 5' probe group 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 NUP214 gene 3' probe group, the ABL1 gene 3' probe group, and the SET gene 5' probe group; Step 2, dividing the non-repetitive genomic sequences into 1 kb blocks respectively, and performing probe design and screening using OligoArray software to obtain a NUP214 gene 3' probe pool, an ABL1 gene 3' probe pool, and a SET gene 5' 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 NUP214 gene 3' probe pool, an ABL1 gene 3' probe pool, and a SET gene 5' probe pool containing tag sequences; Step 4, chemically synthesizing the probes in the NUP214 gene 3' probe pool, the ABL1 gene 3' probe pool and the SET gene 5' probe pool respectively, and mixing them evenly to obtain the NUP214 gene 3' probe library, the ABL1 gene 3' probe library and the SET gene 5' probe library; Step 5: Use universal primers containing different fluorescent groups to perform amplification and labeling reaction on the probes in the NUP214 gene 3' probe library, the ABL1 gene 3' probe library and the SET gene 5' probe library to obtain the NUP214 gene 3' probe group, the ABL1 gene 3' probe group and the SET gene 5' 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 17bp tag sequence at the 5' end is: 5'-TGTAAAACGACGGCCAG-3' (SEQ ID NO.1), and the 18bp 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 different fusion genes of NUP214, 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 genes of NUP214.

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