Primer and kit for detecting CDC42SE2:: HOXA13 fusion gene as well as use method and application

Abstract

The invention relates to the technical field of gene detection, in particular to a primer and a kit for detecting a CDC42SE2:: HOXA13 fusion gene as well as a use method and application of the primer and the kit. The primer comprises a CDC42SE2:: HOXA13 primer probe sequence and an ABL1 internal reference primer probe sequence. Rare CDC42SE2: HOXA13 gene fusion is efficiently detected through a specific primer probe sequence combination, and when CDC42SE2: HOXA13 and ABL1 internal reference primer probe sequences formed by specific exon DNA chains are adopted, target fusion genes and internal reference genes can be accurately captured and recognized. The discovery opens up a brand new way for accurately detecting the CDC42SE2:: HOXA13 fusion gene and optimizing a diagnosis and treatment scheme of mixed phenotype acute leukemia.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and particularly relates to a primer, a kit, a use method and an application for detecting the CDC42SE2::HOXA13 fusion gene. Background Art

[0002] Mixed phenotype acute leukemia (MPAL) is a rare acute leukemia (AL), which is mainly characterized by the co-expression of multiple cell line phenotypes of myeloid, T / B cell lines in leukemia cells. MPAL accounts for about 2.8% (0.3% - 9.0%) of AL. Based on the research results of the SEER (Surveillance, Epidemiology, and End Results) database, the annual incidence of MPAL is 0.35 / 10 6 . MPAL usually has a worse prognosis than acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL). At present, the pathogenesis of MPAL is not clear. The possible pathogenesis is the continuous accumulation of gene mutations produced by multipotent lymphoid progenitors (MPL), resulting in the emergence of a mixed phenotype. Currently, few MPAL fusion genes have been discovered, and there is still a blank for the detection kit for the fusion genes found in MPAL.

[0003] In the prior art, some detection ideas for leukemia-related fusion genes have been proposed. For example, Chinese Patent CN116790785A discloses a probe pool for detecting blood tumor fusion genes, a preparation method and an application thereof. Chinese Patent CN117305457A discloses a blood tumor multi-gene detection kit and a preparation method thereof. Chinese Patent CN115948553A discloses a primer set and a method for detecting blood tumor genes, which can qualitatively detect known, unknown, and common gene fusion sites of multiple genes including the HOXA13 gene through high-throughput sequencing technology, but cannot accurately and quantitatively detect the CDC42SE2::HOXA13 fusion gene. Another example is Chinese Patent CN117757910A, which discloses a leukemia fusion gene single-tube multiplex PCR detection method and a kit thereof, which can detect leukemia-related fusion genes by a method combining multiplex PCR and first-generation sequencing, but this detection method does not include the CDC42SE2::HOXA13 fusion type and cannot quantitatively detect the CDC42SE2::HOXA13 fusion gene.

[0004] In such a background, there is an urgent need to provide a detection method that can specifically monitor rare acute leukemia. Summary of the Invention

[0005] The present invention explores and discovers that there are MPAL patients carrying the CDC42SE2::HOXA13 fusion gene, and this fusion gene is of great significance for the diagnosis, treatment, and monitoring of residual micro-lesions of MPAL disease. Moreover, there is currently no technology to achieve quantitative detection of CDC42SE2::HOXA13. The present invention provides a set of detection primers for the CDC42SE2::HOXA13 fusion gene, and in combination with fluorescence quantitative PCR technology, it can achieve accurate quantitative detection of the CDC42SE2::HOXA13 fusion gene, which has positive research significance for the clinical diagnosis and treatment of rare acute leukemia.

[0006] In the first aspect of the present invention, a primer for detecting the CDC42SE2::HOXA13 fusion gene is provided, and the primer includes the CDC42SE2::HOXA13 primer-probe sequence and the ABL1 internal reference primer-probe sequence.

[0007] In some preferred embodiments, the CDC42SE2::HOXA13 primer-probe sequence includes: CDC42SE2::HOXA13-F: 5’-GGCCAGATTTGAGTGTGTGA-3’

[0008] CDC42SE2::HOXA13-R: 5’-GGGTTCCTGTAGTGGTGATG-3’

[0009] CDC42SE2::HOXA13-P: ’-FAM-CTGAGCCTCGAGCCTAGGAAGG-BHQ1-3’.

[0010] In some preferred embodiments, the ABL1 internal reference primer-probe sequence includes: ABL1 internal reference-F: 5’-AATTGCTACCTATGGCATGT-3’

[0011] ABL1 internal reference-R: 5’-CTTTTCCACTTCGTCTGAGA-3’

[0012] ABL1 internal reference-P: 5’-FAM-GACCTGTCCCAGGTGTATGAGCT-BHQ1-3’.

[0013] The HOXA13 (Homeobox A13) gene is located at 7p15.2 and is a member of the HOX (Homeobox Gene) family A cluster. It can be used as a fusion partner in the detection field of leukemia. The currently discovered fusion partners of HOXA13 include NUP98, GARS1, etc. Through research, it has been found that the expression of HOXA13 is related to the poor prognosis of T-ALL patients. HOXA13-positive patients are more enriched in JAK-STAT pathway mutations, suggesting that clinical detection of HOXA13 expression can help identify T-ALL patients prone to recurrence, and HOXA13-positive patients may benefit from JAK-STAT pathway inhibitor treatment.

[0014] The CDC42SE2 (CDC42 small effector 2) gene is mostly located at 5q31.1 and a small part is located at 5q23.3. This gene can encode a protein consisting of 84 amino acids, and the encoded protein can interact with CDC42 to regulate the function of CDC42 protein. The fusion partners of CDC42SE2 found in diseases such as breast cancer, lung cancer, prostate cancer, and soft tissue sarcoma include ARL15, FBXO38, SPIDR, LYRM7, PLPP1, and BRAF. However, no CDC42SE2-related fusion genes have been found in blood diseases. It has been found that CDC42SE2 can participate in the assembly of the cytoskeleton by interacting with CDC42, and during T cell activation, the CDC42SE2 protein is highly expressed in lymphocytes and immune tissues and is recruited to the immunological synapse at the T cell-APC contact site in an antigen-dependent manner. CDC42SE2 fusion is related to the pathogenesis of MPAL and has the potential to be used as a molecular marker for the diagnosis of MPAL disease.

[0015] The present invention locates the CDC42SE2::HOXA13 fusion gene and provides a specific primer combination for detection. By quantitatively detecting the CDC42SE2::HOXA13 fusion gene, it provides crucial molecular biological basis for the diagnosis of mixed phenotype acute leukemia. Through the precise quantitative analysis of the CDC42SE2::HOXA13 fusion gene, it can provide important biological information basis for the selection of treatment regimens, recurrence risk assessment, and minimal residual disease monitoring of patients, which is of great significance.

[0016] The second aspect of the present invention provides a kit for detecting the CDC42SE2::HOXA13 fusion gene, and the kit includes: nucleic acid amplification reagents, reverse transcription reagents, control products, and reference products.

[0017] The nucleic acid amplification reagent includes a CDC42SE2::HOXA13 PCR reaction solution, an ABL1 internal reference gene PCR reaction solution, and a PCR MIX.

[0018] The CDC42SE2::HOXA13 PCR reaction solution is the above-mentioned CDC42SE2::HOXA13 primer-probe sequence.

[0019] The ABL1 internal reference gene PCR reaction solution is the above-mentioned ABL1 internal reference primer-probe sequence.

[0020] The third aspect of the present invention provides a method for using a kit for detecting the CDC42SE2::HOXA13 fusion gene. The using steps include:

[0021] (1) Extract ribonucleic acid from the sample to be tested, and mix and dissolve the extracted ribonucleic acid with RNase-Free water to obtain a reverse transcription template;

[0022] (2) Place the reverse transcription template and the reverse transcription reagent in a PCR amplifier for mixing to perform a reverse transcription reaction to generate complementary DNA;

[0023] (3) Prepare the amplification reagent and add samples;

[0024] (4) Perform a PCR amplification reaction and detection.

[0025] Preferably, the reverse transcription conditions in step (2) are: reacting at 35-40°C for 0.5-2 h, and then reacting at 60-80°C for 5-30 min to obtain a complementary DNA template.

[0026] Preferably, the reverse transcription conditions in step (2) are: reacting at 37°C for 1 h, and then reacting at 70°C for 10 min to obtain a complementary DNA template.

[0027] In some preferred embodiments, step (3) includes:

[0028] 3-1) Prepare the nucleic acid amplification reagent: respectively mix the CDC42SE2::HOXA13 PCR reaction solution and the ABL1 internal reference gene PCR reaction solution with the PCR MIX, melt at room temperature, vibrate and mix evenly, and centrifuge to obtain the nucleic acid amplification reagent;

[0029] 3-2) Configure the reaction system: Take each complementary DNA template to detect the CDC42SE2::HOXA13 fusion gene and the ABL1 internal reference gene respectively. Therefore, configure the first reaction system and the second reaction system respectively:

[0030] 3-3) Transfer the configured first reaction system and second reaction system to a PCR amplifier for the next treatment.

[0031] Preferably, the first reaction system is specifically as follows: for the CDC42SE2::HOXA13 fusion gene, mix the CDC42SE2::HOXA13 PCR reaction solution, PCR MIX, and complementary DNA template; the volume ratio of the three is (5 - 10):(10 - 15):(3 - 7); preferably 7:13:5.

[0032] Preferably, the second reaction system is specifically as follows: for the ABL1 internal reference gene, mix the ABL1 internal reference gene PCR reaction solution, PCR MIX, and complementary DNA template;; the volume ratio of the three is (5 - 10):(10 - 15):(3 - 7); preferably 7:13:5.

[0033] The fourth aspect of the present invention provides an application of the above-mentioned kit or the use method of the kit in the quantitative detection of the CDC42SE2::HOXA13 fusion gene in patients with mixed phenotype acute leukemia.

[0034] Beneficial effects:

[0035] The present invention provides a primer, a kit, a use method, and an application for detecting the CDC42SE2::HOXA13 fusion gene, having the following advantages:

[0036] (1) The present invention pioneered the discovery that MPAL patients carry a new CDC42SE2::HOXA13 fusion gene, which is a new MPAL-related fusion gene not reported before, and it has been verified that this fusion gene can be used as a molecular marker for MPAL patients, applied to disease diagnosis, treatment plan selection, and minimal residual disease monitoring, providing a new monitoring idea for the clinical diagnosis and treatment of MPAL.

[0037] (2) The present invention efficiently detects the rare CDC42SE2::HOXA13 gene fusion through a specific primer-probe sequence combination. When using the CDC42SE2::HOXA13 and ABL1 internal reference primer-probe sequences composed of specific exon DNA strands, the target fusion gene and the internal reference gene can be accurately captured and identified. This discovery has opened up a brand-new way for accurately detecting the CDC42SE2::HOXA13 fusion gene and optimizing the diagnosis and treatment plan for mixed phenotype acute leukemia.

[0038] (3) The present invention provides a kit capable of directly detecting the CDC42SE2::HOXA13 fusion gene in a sample. This kit integrates reverse transcription reagents, nucleic acid amplification reagents, control products, and reference products. By using the PCR combined with real-time fluorescence probe technology, it realizes the accurate quantitative detection of the RNA content of the CDC42SE2::HOXA13 fusion gene in the sample to be tested, with simple operation and strong practicability.

[0039] (4) The present invention optimizes the usage method of the kit, and accurate results can be quickly obtained by adopting simple detection steps. Verified by multiple experiments, this kit exhibits high specificity, high reproducibility, and high accuracy for the CDC42SE2::HOXA13 fusion gene, and can greatly improve the detection efficiency while ensuring the accuracy of the detection results.

[0040] (5) The detection of the present invention has high sensitivity and can effectively detect samples with as low as 50 copies. It can be used for the diagnosis and treatment of patients with positive CDC42SE2::HOXA13 gene fusion, contributing to the detection of minimal residual lesions in leukemia, which is of great significance for treatment monitoring and treatment plan formulation, and provides strong support for clinical treatment and prognosis evaluation. Description of the Drawings

[0041] Figure 1 . Schematic diagram of the CDC42SE2::HOXA13 fusion gene;

[0042] Figure 2 . Sanger sequencing result diagram of the PCR verification product of patients carrying the fusion gene;

[0043] Figure 3 . Standard quality plasmid map of the CDC42SE2::HOXA13 fusion gene;

[0044] Figure 4 . PCR amplification curves of reference products 1 - 4 of the ABL1 internal reference gene;

[0045] Figure 5 . PCR amplification curves of reference products 1 - 4 of the CDC42SE2::HOXA13 fusion gene;

[0046] Figure 6 . Amplification curve of the second reaction system (ABL1 internal reference gene) during specificity testing;

[0047] Figure 7 . Amplification curve of the first reaction (CDC42SE2::HOXA13 fusion gene) during specificity testing;

[0048] Figure 8 . Amplification curve of the second reaction system (ABL1 internal reference gene) during detection limit verification;

[0049] Figure 9 . Amplification curve of the first reaction (CDC42SE2::HOXA13 fusion gene) during the limit of detection test;

[0050] Figure 10 . Amplification curve of the second reaction system (ABL1 reference gene) for high-copy samples during repeatability verification;

[0051] Figure 11 . Amplification curve of the first reaction (CDC42SE2::HOXA13 fusion gene) for high-copy samples during repeatability testing;

[0052] Figure 12 . Amplification curve of the second reaction system (ABL1 reference gene) for low-copy samples during repeatability verification;

[0053] Figure 13 . Amplification curve of the first reaction (CDC42SE2::HOXA13 fusion gene) for low-copy samples during repeatability testing;

[0054] Figure 14 . Amplification curve of the ABL1 reference gene during clinical sample detection;

[0055] Figure 15 . Amplification curve of the CDC42SE2::HOXA13 fusion gene during clinical sample detection. Detailed implementation manners

[0056] Note: Unless otherwise specified, the solvent of the solutions involved in the present invention is water; and, the raw materials used are all commercially available.

[0057] Example 1

[0058] This example provides a primer, kit and its usage method for detecting the CDC42SE2::HOXA13 fusion gene.

[0059] The primer includes the CDC42SE2::HOXA13 primer-probe sequence and the ABL1 reference primer-probe sequence.

[0060] The CDC42SE2::HOXA13 primer-probe sequence includes:

[0061] CDC42SE2::HOXA13-F: 5’-GGCCAGATTTGAGTGTGTGA-3’

[0062] CDC42SE2::HOXA13-R: 5’-GGGTTCCTGTAGTGGTGATG-3’

[0063] CDC42SE2::HOXA13 - P: 5’-FAM-CTGAGCCTCGAGCCTAGGAAGG-BHQ1-3’。

[0064] The ABL1 internal reference primer - probe sequences include:

[0065] ABL1 internal reference - F: 5’-AATTGCTACCTATGGCATGT-3’

[0066] ABL1 internal reference - R: 5’-CTTTTCCACTTCGTCTGAGA-3’

[0067] ABL1 internal reference - P: 5’-FAM-GACCTGTCCCAGGTGTATGAGCT-BHQ1-3’。

[0068] In the second aspect of this embodiment, a kit for detecting the CDC42SE2::HOXA13 fusion gene is provided; the kit includes: nucleic acid amplification reagents, reverse transcription reagents, control products and reference products.

[0069] The nucleic acid amplification reagents, reverse transcription reagents, control products and reference products all come from Shanghai Yuanqi Biomedical Technology Co., Ltd.

[0070] The nucleic acid amplification reagents include CDC42SE2::HOXA13 PCR reaction solution, ABL1 internal reference gene PCR reaction solution and PCR MIX.

[0071] The CDC42SE2::HOXA13 PCR reaction solution is the above - mentioned CDC42SE2::HOXA13 primer - probe sequence.

[0072] The ABL1 internal reference gene PCR reaction solution is the above - mentioned ABL1 internal reference primer - probe sequence.

[0073] The reverse transcription reagent consists of MIX 1 and MIX 2; the volume ratio of MIX 1 to MIX 2 is 3:2, with a total of 10 μL.

[0074] Among them, MIX1 is specifically: dNTP (10 mM), random primers (100 μM).

[0075] Among them, MIX2 is specifically: RNase (10 U), reverse transcriptase (200 U).

[0076] In the third aspect of this embodiment, a method for using the above - mentioned kit is provided, and the using steps include:

[0077] (1) Treatment of the sample to be tested: Extract ribonucleic acid (RNA) from the sample to be tested, and mix and dissolve the extracted ribonucleic acid with RNase-Free water to obtain a reverse transcription template;

[0078] (2) Reverse transcription of complementary DNA;

[0079] (3) Prepare amplification reagents and load samples;

[0080] (4) PCR amplification reaction and detection.

[0081] In step (1), the Trizol method is used to extract RNA from the sample to be tested. During detection, the extracted RNA precipitate is mixed and dissolved with RNase-Free water (the volume of RNase-Free water is 50 μL), and 10 μL of the mixed solution is taken as the reverse transcription template; at the same time, a negative control sample is prepared, and the negative control sample is specifically RNase-Free water of the same volume. The selection of the sample to be tested is specifically described in the test method.

[0082] Step (2) is specifically as follows: The reverse transcription template obtained in step (1) and the reverse transcription reagent are placed in a PCR amplifier for mixing to perform reverse transcription to synthesize complementary DNA; the reverse transcription conditions are: reacting at 37 °C for 1 h, and then reacting at 70 °C for 10 min to obtain a complementary DNA template.

[0083] Step (3) includes:

[0084] 3-1) Prepare nucleic acid amplification reagents: Mix the CDC42SE2::HOXA13 PCR reaction solution and the ABL1 internal reference gene PCR reaction solution with PCR MIX respectively (volume ratio is 7:13), melt at room temperature and mix well by vibration, and centrifuge at 2000 rpm for 10 s to obtain nucleic acid amplification reagents;

[0085] 3-2) Configure the reaction system: Each complementary DNA template is used to detect the CDC42SE2::HOXA13 fusion gene and the ABL1 internal reference gene respectively, so the first reaction system and the second reaction system are configured respectively:

[0086] The first reaction system is specifically: For the CDC42SE2::HOXA13 fusion gene, mix the CDC42SE2::HOXA13 PCR reaction solution, PCR MIX and the complementary DNA template; the volume ratio of the three is 7:13:5, and the total volume of the first reaction system is 25 μL.

[0087] The second reaction system is specifically: For the ABL1 internal reference gene, mix the ABL1 internal reference gene PCR reaction solution, PCR MIX and the complementary DNA template; the volume ratio of the three is 7:13:5, and the total volume of the second reaction system is 25 μL.

[0088] Step 3-3) Transfer the prepared first reaction system and second reaction system to a PCR amplifier for the next treatment.

[0089] The specific conditions for PCR amplification in step (4) are as follows: maintain at 42°C for 5 min; then raise to 94°C and maintain for 3 min; ② PCR cycling stage: maintain at 94°C for 15 s; maintain at 60°C for 60 s; the number of cycles is 40 times. After the cycling is completed, collect the fluorescence signal.

[0090] Note: When using a PCR amplifier for testing, the detection channel is set to: FAM, and the reference fluorescence is set to none.

[0091] The detection in step (4) is realized by the software supporting the PCR amplifier.

[0092] The PCR amplifier used in this kit is the Bio-Rad CFX96 fluorescence PCR instrument.

[0093] Performance test

[0094] 1. Fusion gene verification

[0095] Applying bioinformatics technology, by analyzing the sample data of MPAL patients (from Shengjing Hospital Affiliated to China Medical University) obtained by high-throughput sequencing, the existence of the CDC42SE2::HOXA13 fusion gene was found, and this fusion gene was not included in the relevant fusion gene databases. Further, by designing sequencing primers and using first-generation sequencing to verify the fusion result, it was found that the specific gene fusion form was the fusion of exon 1 of CDC42SE2 and partial exon 2 of HOXA13, forming the CDC42SE2::HOXA13 fusion gene. See the schematic diagram of the fusion gene in Figure 1 .

[0096] Perform PCR amplification on the fusion gene and conduct Sanger sequencing on the PCR amplification product to determine that the MPAL patient carries the novel CDC42SE2::HOXA13 fusion gene. See the verification result of Sanger sequencing in Figure 2 .

[0097] According to the Sanger sequencing result, select a sequence containing the break point and clone it into a plasmid (see the plasmid map in Figure 3 , from Sangon Biotech (Shanghai) Co., Ltd.). After verification by Sanger sequencing, it is determined that the plasmid sequence of the CDC42SE2::HOXA13 fusion gene is correct, and the cloned fusion gene plasmid is selected for preparing the linear standard.

[0098] The determined sequence of the CDC42SE2::HOXA13 fusion gene plasmid (3089bp) is as follows:

[0099]

[0100] 2. Method effectiveness determination

[0101] Perform PCR detection on the linear standard curves of the CDC42SE2::HOXA13 fusion gene and the ABL1 internal reference gene (refer to steps 2-4 in the kit usage method of Example 1, and the information of each reference product is shown in Table 1 below).

[0102] Table 1

[0103] Reference product name Reference product concentration CDC42SE2::HOXA13 fusion gene linear standard 1 <![CDATA[1.0×10 6 copies]]> CDC42SE2::HOXA13 fusion gene linear standard 2 <![CDATA[1.0×10 5 copies]]> CDC42SE2::HOXA13 fusion gene linear standard 3 <![CDATA[1.0×10 4 copies]]> CDC42SE2::HOXA13 fusion gene linear standard 4 <![CDATA[1.0×10 3 copies]]> ABL1 endogenous reference gene linear standard 1 <![CDATA[1.0×10 6 copies]]> ABL1 endogenous reference gene linear standard 2 <![CDATA[1.0×10 5 copies]]> ABL1 endogenous reference gene linear standard 3 <![CDATA[1.0×10 4 copies]]> ABL1 endogenous reference gene linear standard 4 <![CDATA[1.0×10 3 copies]]>

[0104] The C values of the linear standards 1-4 of the CDC42SE2::HOXA13 fusion gene and the ABL1 internal reference gene should all be ≤ 36, and the absolute value of the standard curve fitting degree should be ≥ 0.980. The C value of the negative control should be ≥ 38 or show "Undet". The test results of the final reference product and the negative control meet the above requirements. T The C value of the negative control should be ≥ 38 or show "Undet". The test results of the final reference product and the negative control meet the above requirements. T The C value of the negative control should be ≥ 38 or show "Undet". The test results of the final reference product and the negative control meet the above requirements.

[0105] 3. Qualitative determination:

[0106] Perform qualitative determination according to the C value of the reaction solution, and the determination basis is shown in Table 2 below. T Perform qualitative determination according to the C value of the reaction solution, and the determination basis is shown in Table 2 below.

[0107] Table 2

[0108]

[0109]

[0110] If after re-detection, the C value of the CDC42SE2::HOXA13 PCR reaction solution < 38, then the CDC42SE2::HOXA13 fusion gene is positive; if the C value of the CDC42SE2::HOXA13 PCR reaction solution ≥ 38, then the CDC42SE2::HOXA13 fusion gene RNA is below the lowest detection limit. T If after re-detection, the C value of the CDC42SE2::HOXA13 PCR reaction solution < 38, then the CDC42SE2::HOXA13 fusion gene is positive; if the C value of the CDC42SE2::HOXA13 PCR reaction solution ≥ 38, then the CDC42SE2::HOXA13 fusion gene RNA is below the lowest detection limit. T If after re-detection, the C value of the CDC42SE2::HOXA13 PCR reaction solution < 38, then the CDC42SE2::HOXA13 fusion gene is positive; if the C value of the CDC42SE2::HOXA13 PCR reaction solution ≥ 38, then the CDC42SE2::HOXA13 fusion gene RNA is below the lowest detection limit.

[0111] For samples with the C value of the internal reference reaction solution > 36 and the C value of the CDC42SE2::HOXA13 PCR reaction solution > 36, the sampling amount of this sample needs to be increased, and after re-extracting RNA, perform PCR detection (that is, repeat steps S1-S5 after increasing the sampling amount), and the test results are determined with reference to Table 1. If there is still a situation where the C value of the internal reference reaction solution > 36 and the C value of the CDC42SE2::HOXA13 reaction solution > 36, then it is determined that the sample does not meet the requirements. T For samples with the C value of the internal reference reaction solution > 36 and the C value of the CDC42SE2::HOXA13 PCR reaction solution > 36, the sampling amount of this sample needs to be increased, and after re-extracting RNA, perform PCR detection (that is, repeat steps S1-S5 after increasing the sampling amount), and the test results are determined with reference to Table 1. If there is still a situation where the C value of the internal reference reaction solution > 36 and the C value of the CDC42SE2::HOXA13 reaction solution > 36, then it is determined that the sample does not meet the requirements. T For samples with the C value of the internal reference reaction solution > 36 and the C value of the CDC42SE2::HOXA13 PCR reaction solution > 36, the sampling amount of this sample needs to be increased, and after re-extracting RNA, perform PCR detection (that is, repeat steps S1-S5 after increasing the sampling amount), and the test results are determined with reference to Table 1. If there is still a situation where the C value of the internal reference reaction solution > 36 and the C value of the CDC42SE2::HOXA13 reaction solution > 36, then it is determined that the sample does not meet the requirements. T For samples with the C value of the internal reference reaction solution > 36 and the C value of the CDC42SE2::HOXA13 PCR reaction solution > 36, the sampling amount of this sample needs to be increased, and after re-extracting RNA, perform PCR detection (that is, repeat steps S1-S5 after increasing the sampling amount), and the test results are determined with reference to Table 1. If there is still a situation where the C value of the internal reference reaction solution > 36 and the C value of the CDC42SE2::HOXA13 reaction solution > 36, then it is determined that the sample does not meet the requirements. T For samples with the C value of the internal reference reaction solution > 36 and the C value of the CDC42SE2::HOXA13 PCR reaction solution > 36, the sampling amount of this sample needs to be increased, and after re-extracting RNA, perform PCR detection (that is, repeat steps S1-S5 after increasing the sampling amount), and the test results are determined with reference to Table 1. If there is still a situation where the C value of the internal reference reaction solution > 36 and the C value of the CDC42SE2::HOXA13 reaction solution > 36, then it is determined that the sample does not meet the requirements.

[0112] 4. Quantitative determination:

[0113] 1) Draw a standard curve: Take linear standards 1-4 of the CDC42SE2::HOXA13 fusion gene and the ABL1 internal reference gene for PCR detection (refer to steps S2-S4 in the kit usage method of Example 1). Specifically, add linear standards 1-4 to the two reaction systems in step S3 according to the addition amounts in Table 1 for determination.

[0114] Among them, the addition amounts of linear standards 1-4 of the CDC42SE2::HOXA13 fusion gene in the first reaction system (CDC42SE2::HOXA13 fusion gene) are set as: 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 copies. The addition amounts of linear standards 1-4 of the ABL1 internal reference gene in the second reaction system (ABL1 internal reference gene) are set as: 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 copies; After the PCR amplification reaction is completed, the standard curve can be drawn according to the addition amounts of linear standards 1-4 and the fluorescence signal test results.

[0115] 2) Substitute the test results of the samples into the two standard curves in step 1) to obtain the detected concentrations (A) of the CDC42SE2::HOXA13 fusion gene and the detected concentrations (B) of the ABL1 internal reference gene for each sample respectively.

[0116] 3) Analysis of test results:

[0117] a. If the detected concentration (A) of the CDC42SE2::HOXA13 fusion gene > 1×10 7 copies, that is, it is not within the linear range, it needs to be appropriately diluted as appropriate and retested.

[0118] b. If the detected concentration (B) of the ABL1 internal reference gene > 1×10 7 copies, that is, it is not within the linear range, it needs to be appropriately diluted as appropriate and retested.

[0119] c. If the test result is 10 copies ≤ the detected concentration (A) of the CDC42SE2::HOXA13 fusion gene ≤ 1×10 7 copies, and 1×10 3 copies ≤ the detected concentration (B) of the ABL1 internal reference gene ≤ 1×10 7If there are copies, the output fusion gene ratio is the ratio of the detection concentration of the CDC42SE2::HOXA13 fusion gene (A) to the detection concentration of the ABL1 internal reference gene (B) (i.e., A / B) × 100%.

[0120] Among them, the PCR amplification curves of the ABL1 internal reference gene linear standards 1-4 are shown in Figure 4 ; the corresponding standard curve parameters are: E = 102.1%, R 2 = 100%, Slope = -3.274, y-int = 40.783.

[0121] The PCR amplification curves of the CDC42SE2::HOXA13 fusion gene linear standards 1-4 are shown in Figure 5 ; the corresponding standard curve parameters are: E = 100.9%, R 2 = 99.8%, Slope = -3.300, y-int = 39.944.

[0122] 5. Specificity test verification

[0123] Take 35 CDC42SE2::HOXA13 negative samples (from Shengjing Hospital Affiliated to China Medical University), and perform PCR detection according to the S1-S4 steps of the kit usage method in Example 1 to verify the specificity of this kit; the detection results are shown in Figure 6 and Figure 7 .

[0124] Among them, the test results of each sample of the ABL1 internal reference gene reaction solution are shown in Table 3 below.

[0125] Table 3

[0126]

[0127]

[0128] Among them, the test results of each sample of the CDC42SE2::HOXA13 fusion gene reaction solution are shown in Table 4 below.

[0129] (N / A: No signal detected)

[0130] Table 4

[0131]

[0132]

[0133] Based on the test results in Table 3 and Table 4, it can be seen that the detection results of the ABL1 internal reference gene in 35 samples were normal, and no signals were detected for the CDC42SE2::HOXA13 fusion gene. The above results indicate that the kit has good specificity.

[0134] 6. Verification of the detection limit

[0135] By performing PCR detection on the detection limit reference sample (a sample with 50 copies of CDC42SE2::HOXA13) (referring to steps S3 - S4 in the kit usage method in Example 1), it was verified whether the kit of the present invention could achieve 20 detections with a detection rate ≥ 95%. According to the test results in Table 5, all 20 detections of the detection limit reference sample using the kit of the present invention were positive, with a detection rate of 100%. This indicates that the kit of the present invention can detect samples with a minimum of 50 copies of CDC42SE2::HOXA13; the PCR test curve is shown in Figure 8 、 Figure 9 。

[0136] Table 5

[0137]

[0138] 7. Repeatability:

[0139] The reproducibility of the detection results of the ABL1 internal reference gene and the CDC42SE2::HOXA13 fusion gene was investigated respectively.

[0140] ABL1 internal reference gene: The same high-copy sample and the same low-copy sample (specifically, the RNA extracted from pseudovirus) were repeatedly detected 10 times (corresponding to the second reaction system), and the PCR test curve is shown in Figure 10 、 Figure 12 。 The 10 test results were recorded in Table 5. The calculated CV values of the ABL1 internal reference gene copy number results corresponding to the corresponding order of magnitude for the high-copy sample and the low-copy sample were 0.24% and 0.48% respectively, indicating that the detection kit has good repeatability for the detection results of the ABL1 internal reference gene determination.

[0141] CDC42SE2::HOXA13 fusion gene: The same high-copy sample and the same low-copy sample (specifically, the RNA extracted from pseudovirus) were repeatedly detected 10 times (corresponding to the first reaction system), and the PCR test curve is shown in Figure 11 、 Figure 13The results of 10 tests were recorded in Table 6. The CV values of high-copy and low-copy samples corresponding to the copy number results of the CDC42SE2::HOXA13 fusion gene were 1.02% and 0.87% respectively, indicating that the detection results of this detection kit for the CDC42SE2::HOXA13 fusion gene had good repeatability.

[0142] Table 6

[0143]

[0144]

[0145] 6. Clinical sample testing

[0146] According to the kit usage method in Example 1, for 1 positive sample from Shengjing Hospital Affiliated to China Medical University (the fusion gene ratio was calculated according to the ratio of the detection concentration of the CDC42SE2::HOXA13 fusion gene to the detection concentration of the ABL1 internal reference gene; the PCR amplification curves of the positive sample are shown in Figure 14 、 Figure 15 , and the test results of the positive sample are shown in Table 7 below.

[0147] Table 7

[0148]

Claims

1. A primer for detecting the CDC42SE2::HOXA13 fusion gene, characterized in that, The primers include the CDC42SE2::HOXA13 primer-probe sequence and the ABL1 internal reference primer-probe sequence.

2. The primer for detecting the CDC42SE2::HOXA13 fusion gene according to claim 1, characterized in that, The CDC42SE2::HOXA13 primer-probe sequence includes: CDC42SE2::HOXA13-F: 5’-GGCCAGATTTGAGTGTGTGA-3’.

3. The primer for detecting the CDC42SE2::HOXA13 fusion gene according to claim 2, characterized in that, The CDC42SE2::HOXA13 primer-probe sequence further includes: CDC42SE2::HOXA13-R: 5’-GGGTTCCTGTAGTGGTGATG-3’.

4. The primer for detecting the CDC42SE2::HOXA13 fusion gene according to claim 3, wherein The CDC42SE2::HOXA13 primer-probe sequence further includes: CDC42SE2::HOXA13-P: 5’-FAM-CTGAGCCTCGAGCCTAGGAAGG-BHQ1-3’.

5. A kit for detecting the CDC42SE2::HOXA13 fusion gene according to any one of claims 1-4, characterized in that, The kit includes: nucleic acid amplification reagents, reverse transcription reagents, control products and reference products; The nucleic acid amplification reagents include the CDC42SE2::HOXA13 PCR reaction solution, the ABL1 internal reference gene PCR reaction solution and PCRMIX; The CDC42SE2::HOXA13 PCR reaction solution is the CDC42SE2::HOXA13 primer-probe sequence described in claim 1; The ABL1 internal reference gene PCR reaction solution is the ABL1 internal reference primer-probe sequence described in claim 1.

6. A method for using a kit for detecting the CDC42SE2::HOXA13 fusion gene according to claim 5, characterized in that, The using steps include: (1) Extract ribonucleic acid from the sample to be tested, and mix and dissolve the extracted ribonucleic acid with RNase-Free water to obtain a reverse transcription template; (2) Place the reverse transcription template and the reverse transcription reagents in a PCR amplifier for mixing to perform a reverse transcription reaction to generate complementary DNA; (3) Prepare amplification reagents and load the samples; (4) Perform PCR amplification reaction and detection.

7. The primer for detecting the CDC42SE2::HOXA13 fusion gene according to claim 6, wherein The reverse transcription conditions in step (2) are: react at 35 - 40 °C for 0.5 - 2 h, and then react at 60 - 80 °C for 5 - 30 min to obtain a complementary DNA template.

8. The primer for detecting the CDC42SE2::HOXA13 fusion gene according to claim 6, characterized in that, Step (3) includes: 3-1) Prepare nucleic acid amplification reagents: respectively mix the CDC42SE2::HOXA13 PCR reaction solution and the ABL1 internal reference gene PCR reaction solution with PCR MIX, melt at room temperature, vibrate and mix evenly, and centrifuge to obtain nucleic acid amplification reagents; 3-2) Configure the reaction system: Take each complementary DNA template to detect the CDC42SE2::HOXA13 fusion gene and the ABL1 internal reference gene respectively, so configure the first reaction system and the second reaction system respectively: 3-3) Transfer the configured first reaction system and second reaction system to a PCR amplifier for the next treatment.

9. The primer for detecting the CDC42SE2::HOXA13 fusion gene according to claim 8, wherein The first reaction system is specifically: for the CDC42SE2::HOXA13 fusion gene, mix the CDC42SE2::HOXA13 PCR reaction solution, PCR MIX and the complementary DNA template; the volume ratio of the three is (5 - 10):(10 - 15):(3 - 7); The specific second reaction system is as follows: for the ABL1 internal reference gene, mix the ABL1 internal reference gene PCR reaction solution, PCR MIX, and complementary DNA template; the volume ratio of the three is (5-10):(10-15):(3-7).

10. An application of the kit according to claim 5 or the use method of the kit according to claim 6 in the quantitative detection of the CDC42SE2::HOXA13 fusion gene in patients with mixed phenotype acute leukemia.

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