A primer-probe combination, kit and application for detecting RARG fusion gene

Abstract

The present invention discloses a primer-probe combination, a kit and an application for detecting the RARG fusion gene. The primer-probe combination for detecting the RARG fusion gene disclosed by the present invention includes primers 1-12 and probes 1-3. The primers 1-12 are 12 single-stranded DNAs shown in SEQ ID No.1-12 in the sequence listing respectively, and the sequences of the probes 1-3 are SEQ ID No.13, 14 and 15 in the sequence listing respectively. The primer-probe combination of the present invention has the following characteristics: comprehensive coverage: the primer-probe combination for detecting the RARG fusion gene of the present invention can detect 7 types of RARG fusion genes; accurate and reliable: the primer-probe combination for detecting the RARG fusion gene of the present invention can specifically detect the RARG fusion gene and its 7 fusion types; simple, rapid and low-cost in operation. The present invention has good application prospects.

Description

Technical Field

[0001] The present invention relates to a primer-probe combination, a kit and an application for detecting RARG fusion gene in the field of biomedicine. Background Art

[0002] Acute myeloid leukemia (AML) is a type of hematological malignancy originating from hematopoietic stem / progenitor cells, accounting for approximately 80% of adult acute leukemias. Current treatment methods mainly include chemotherapy and hematopoietic stem cell transplantation (HSCT). The latest SEER data in the United States shows that the 5-year survival rate of AML is only 29.5%. AML is a major disease seriously threatening people's health. Therefore, the precise diagnosis and treatment of AML have become the focus of international research. AML is a group of highly heterogeneous diseases. According to the results of molecular genetics, the European LeukemiaNet (ELN) and the National Comprehensive Cancer Network (NCCN) guidelines in the United States classify AML into three groups with good, intermediate, and poor prognoses. The molecular typing diagnosis and treatment strategy based on this has successfully improved the prognosis of some patients. With the emergence and rapid application of next-generation sequencing technology, more and more molecular abnormalities have been discovered, and more AML subtypes have been recognized. So far, according to the latest 4th edition of the "WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues", 19 AML subtypes have been defined, which has become the most authoritative typing standard in this field internationally.

[0003] However, the WHO classification does not solve all diagnostic typing problems. It is an open system and awaits the discovery and supplementation of new types. In recent years, the applicant has found a special type of AML patients. Their clinical manifestations, coagulation tests, bone marrow morphology, and immunophenotyping are very similar to those of acute promyelocytic leukemia (APL). Molecular genetic testing shows no t(15;17) / PML-RARA and other RARA rearrangements, and they are ineffective in treatment with retinoic acid (ATRA) and arsenic trioxide (ATO). Such patients are not described in the WHO classification and cannot be typed. Clinicians usually generally diagnose them as AML for treatment. Later, through scientific research means using the RNA-seq method, it was found that such patients have a fusion of retinoic acid receptor gamma (RARG) and other genes. And with the popularization of the RNA-seq method and the decrease in detection costs, more and more cases have been discovered. The applicant defines this type of leukemia as RARG rearrangement acute myeloid leukemia (abbreviated as RARG-AML).

[0004] From the existing data, it is difficult to distinguish RARG-AML from APL based on clinical manifestations and routine examination results. Clinically, patients with RARG-AML also mainly present with coagulation abnormalities accompanied by primary fibrinolysis hyperactivity. Decrease in two or three cell lines is common. In bone marrow morphology, abnormal promyelocytes with increased granules are mainly seen and POX is strongly positive. Immunophenotyping shows the characteristics of typical APL, CD34-HLA-DR-CD11b-CD117+CD13+CD33+MPO+. From the perspective of rapid diagnosis, FISH method is of great significance. If the 17q abnormality is not detected in patients suspected of APL, classical and variant APL can be basically excluded, and RNA-Seq sequencing needs to be initiated in a timely manner. However, since current RNA-Seq sequencing is mainly for scientific research, the detection cost is relatively high, and the reporting time of detection results is relatively long (more than 2-3 weeks), which limits its clinical popularization and application, resulting in the missed diagnosis or misdiagnosis of most RARG-AML cases. From the published RARG-AML cases, the average time from initial diagnosis to definite diagnosis is 50 days (19 days - 32 months). Therefore, there is an urgent need to develop a set of rapid multiplex PCR kits to rapidly screen various RARG fusion genes.

[0005] The clinical significance of rapid screening of RARG fusion genes is also reflected in: 1) Guiding clinical medication: Since RARG-AML is resistant to both retinoic acid and arsenic trioxide, if accurate diagnosis cannot be made, it will lead to the aggravation of the disease and even death. Once RARG-AML is diagnosed, the treatment plan should be immediately changed to the standard chemotherapy plan (DA plan, IA plan, HA plan) to enable the patient to achieve remission and receive subsequent treatment. 2) Providing minimal residual disease (MRD) monitoring indicators: Fusion genes are unique to leukemia cells and are ideal MRD monitoring molecular markers, which can specifically and sensitively reflect the leukemia burden and guide clinical intervention measures as early as possible to prevent recurrence. Therefore, rapid screening of RARG fusion genes is of great clinical significance.

[0006] Transcriptome sequencing technology can be used for fusion gene screening. Although it has advantages such as comprehensive coverage, there are still problems such as cumbersome and complex analysis processes and time-consuming in its application to clinical routine, so it has more advantages in finding unknown types. In contrast, the multiplex RQ-PCR technology combining real-time quantitative PCR (RQ-PCR) and multiplex PCR is a detection technology suitable for screening specific fusion gene types in clinical routine.

[0007] Literature reports and the applicant's previous work show that a total of 35 cases of RARG-AML have been reported internationally, and there are 7 types of RARG fusion genes (NUP98-RARG, PML-RARG, CPSF6-RARG, HNRNPC-RARG, HNRNPM-RARG, NPM1-RARG, SART3-RARG). However, the incidence rates among different types vary greatly. SUMMARY OF THE INVENTION

[0008] The technical problem to be solved by the present invention is how to detect the RARG fusion gene and how to screen RARG-AML patients.

[0009] To solve the above technical problem, the present invention first provides a primer-probe combination for detecting the RARG fusion gene. The primer-probe combination includes primers 1-12 and probes 1-3. The primers 1-12 are 12 single-stranded DNAs shown in SEQ ID No.1-12 in the sequence listing, and the sequences of the probes 1-3 are SEQ ID No.13, 14 and 15 in the sequence listing respectively.

[0010] In the above primer-probe combination, both ends of the probes 1-3 can be respectively labeled with a fluorescent group (such as FAM) and a fluorescence quenching group (such as TAMRA).

[0011] The above primer-probe combination may further include primers and probes for an internal reference. The primers for the internal reference may be primers 13-14. The primers 13-14 are 2 single-stranded DNAs shown in SEQ ID No.16-17 in the sequence listing respectively. The probe may be probe 4, and the sequence of the probe 4 is SEQ ID No.18 in the sequence listing.

[0012] Both ends of the probe 4 can be respectively labeled with a fluorescent group (such as FAM) and a fluorescence quenching group (such as TAMRA).

[0013] The above primer-probe combination may consist only of the primers 1-12 and the probes 1-3, or may also consist of the primers 1-12, the probes 1-3, the primers 13-14 and the probe 4.

[0014] In the above primer-probe combination, each primer and probe can be independently packaged or packaged together. In the above primer-probe combination, the molar amounts of each primer can be equal, the molar amounts of each probe can be equal, and the molar ratio of each primer to each probe can be 3:2.

[0015] The present invention also provides a kit for detecting the RARG fusion gene, and the kit contains the primer-probe combination.

[0016] The present invention also provides any one of the following applications of the primer-probe combination or the kit:

[0017] X1) Detecting or assisting in detecting the RARG fusion gene;

[0018] X2) Preparing a product for detecting or assisting in detecting the RARG fusion gene;

[0019] X3) Preparation of a product for screening or assisting in the screening of RARG-AML patients;

[0020] X4) Preparation of a product for screening or assisting in the screening of non-RARG-AML patients;

[0021] X5) Preparation of a product for diagnosing or assisting in the diagnosis of RARG-AML patients.

[0022] Among them, the detection of the RARG fusion gene can be for non-diagnostic purposes.

[0023] The RARG fusion gene is CPSF6-RARG (i.e., the fusion gene of the CPSF6 gene and the RARG gene), HNRNPC-RARG (i.e., the fusion gene of the HNRNPC gene and the RARG gene), HNRNPM-RARG (i.e., the fusion gene of the HNRNPM gene and the RARG gene), NPM1-RARG (i.e., the fusion gene of the NPM1 gene and the RARG gene), NUP98-RARG (i.e., the fusion gene of the NUP98 gene and the RARG gene), PML-RARG (i.e., the fusion gene of the PML gene and the RARG gene) and / or SART3-RARG (i.e., the fusion gene of the SART3 gene and the RARG gene).

[0024] The present invention also provides any of the following primer-probe combinations:

[0025] P1) A primer-probe combination for detecting the CPSF6-RARG fusion gene, including primers 1-2, 10-12 and probe 1-3. The primers 1-2, 10-12 are respectively 5 single-stranded DNAs shown in SEQ ID No.1-2, 10-12 in the sequence listing, and the sequences of the probes 1-3 are respectively SEQ ID No.13, 14 and 15 in the sequence listing;

[0026] P2) A primer-probe combination for detecting the HNRNPC-RARG fusion gene, including primers 3-4, 10-12 and probe 1-3. The primers 3-4, 10-12 are respectively 5 single-stranded DNAs shown in SEQ ID No.3-4, 10-12 in the sequence listing, and the sequences of the probes 1-3 are respectively SEQ ID No.13, 14 and 15 in the sequence listing;

[0027] P3) A primer-probe combination for detecting the HNRNPM-RARG fusion gene, including primer 5, 10-12 and probe 1-3. The primer 5, 10-12 are respectively 4 single-stranded DNAs shown in SEQ ID No.5, 10-12 in the sequence listing, and the sequences of the probes 1-3 are respectively SEQ ID No.13, 14 and 15 in the sequence listing;

[0028] P4) A primer-probe combination for detecting the NPM1-RARG fusion gene, comprising primers 6, 10-12 and probes 1-3. The primers 6, 10-12 are 4 single-stranded DNAs shown as SEQ ID No.6, 10-12 in the sequence listing respectively, and the sequences of the probes 1-3 are SEQ ID No.13, 14 and 15 in the sequence listing respectively;

[0029] P5) A primer-probe combination for detecting the NUP98-RARG fusion gene, comprising primers 7, 10-12 and probes 1-3. The primers 7, 10-12 are 4 single-stranded DNAs shown as SEQ ID No.7, 10-12 in the sequence listing respectively, and the sequences of the probes 1-3 are SEQ ID No.13, 14 and 15 in the sequence listing respectively;

[0030] P6) A primer-probe combination for detecting the PML-RARG fusion gene, comprising primers 8, 10-12 and probes 1-3. The primers 8, 10-12 are 4 single-stranded DNAs shown as SEQ ID No.8, 10-12 in the sequence listing respectively, and the sequences of the probes 1-3 are SEQ ID No.13, 14 and 15 in the sequence listing respectively;

[0031] P7) A primer-probe combination for detecting the SART3-RARG fusion gene, comprising primers 9, 10-12 and probes 1-3. The primers 9, 10-12 are 4 single-stranded DNAs shown as SEQ ID No.9, 10-12 in the sequence listing respectively, and the sequences of the probes 1-3 are SEQ ID No.13, 14 and 15 in the sequence listing respectively.

[0032] Wherein, both ends of the probes 1-3 can be respectively labeled with a fluorescent group (such as FAM) and a fluorescence quenching group (such as TAMRA).

[0033] The present invention also provides any one of the following kits:

[0034] Q1) A kit for detecting the CPSF6-RARG fusion gene, containing the primer-probe combination for detecting the CPSF6-RARG fusion gene;

[0035] Q2) A kit for detecting the HNRNPC-RARG fusion gene, containing the primer-probe combination for detecting the HNRNPC-RARG fusion gene;

[0036] Q3) A kit for detecting the HNRNPM-RARG fusion gene, containing the primer-probe combination for detecting the HNRNPM-RARG fusion gene;

[0037] Q4) A kit for detecting the NPM1-RARG fusion gene, comprising the primer-probe combination for detecting the NPM1-RARG fusion gene;

[0038] Q5) A kit for detecting the NUP98-RARG fusion gene, comprising the primer-probe combination for detecting the NUP98-RARG fusion gene;

[0039] Q6) A kit for detecting the PML-RARG fusion gene, comprising the primer-probe combination for detecting the PML-RARG fusion gene;

[0040] Q7) A kit for detecting the SART3-RARG fusion gene, comprising the primer-probe combination for detecting the SART3-RARG fusion gene.

[0041] The present invention also provides any one of the following applications R1)-R7):

[0042] R1) The application of the primer-probe combination for detecting the CPSF6-RARG fusion gene or the kit for detecting the CPSF6-RARG fusion gene in the detection or auxiliary detection of RARG fusion gene for non-diagnostic purposes, or in the preparation of products for detecting or auxiliary detecting RARG fusion gene, or in the preparation of products for screening or auxiliary screening of RARG-AML patients, or in the preparation of products for diagnosing or auxiliary diagnosing RARG-AML patients, or in the preparation of products for screening or auxiliary screening of RARG-AML patients containing the CPSF6-RARG fusion gene, or in the preparation of products for diagnosing or auxiliary diagnosing RARG-AML patients containing the CPSF6-RARG fusion gene;

[0043] R2) The application of the primer-probe combination for detecting the HNRNPC-RARG fusion gene or the kit for detecting the HNRNPC-RARG fusion gene in the detection or auxiliary detection of RARG fusion gene for non-diagnostic purposes, or in the preparation of products for detecting or auxiliary detecting RARG fusion gene, or in the preparation of products for screening or auxiliary screening of RARG-AML patients, or in the preparation of products for diagnosing or auxiliary diagnosing RARG-AML patients, or in the preparation of products for screening or auxiliary screening of RARG-AML patients containing the HNRNPC-RARG fusion gene, or in the preparation of products for diagnosing or auxiliary diagnosing RARG-AML patients containing the HNRNPC-RARG fusion gene;

[0044] R3) Use of the primer-probe combination for detecting the HNRNPM-RARG fusion gene or the kit for detecting the HNRNPM-RARG fusion gene in the detection or auxiliary detection of the RARG fusion gene for non-diagnostic purposes, or in the preparation of a product for detecting or auxiliary detecting the RARG fusion gene, or in the preparation of a product for screening or auxiliary screening of RARG-AML patients, or in the preparation of a product for diagnosing or auxiliary diagnosing RARG-AML patients, or in the preparation of a product for screening or auxiliary screening of RARG-AML patients containing the HNRNPM-RARG fusion gene, or in the preparation of a product for diagnosing or auxiliary diagnosing RARG-AML patients containing the HNRNPM-RARG fusion gene;

[0045] R4) Use of the primer-probe combination for detecting the NPM1-RARG fusion gene or the kit for detecting the NPM1-RARG fusion gene in the detection or auxiliary detection of the RARG fusion gene for non-diagnostic purposes, or in the preparation of a product for detecting or auxiliary detecting the RARG fusion gene, or in the preparation of a product for screening or auxiliary screening of RARG-AML patients, or in the preparation of a product for diagnosing or auxiliary diagnosing RARG-AML patients, or in the preparation of a product for screening or auxiliary screening of RARG-AML patients containing the NPM1-RARG fusion gene, or in the preparation of a product for diagnosing or auxiliary diagnosing RARG-AML patients containing the NPM1-RARG fusion gene;

[0046] R5) Use of the primer-probe combination for detecting the NUP98-RARG fusion gene or the kit for detecting the NUP98-RARG fusion gene in the detection or auxiliary detection of the RARG fusion gene for non-diagnostic purposes, or in the preparation of a product for detecting or auxiliary detecting the RARG fusion gene, or in the preparation of a product for screening or auxiliary screening of RARG-AML patients, or in the preparation of a product for diagnosing or auxiliary diagnosing RARG-AML patients, or in the preparation of a product for screening or auxiliary screening of RARG-AML patients containing the NUP98-RARG fusion gene, or in the preparation of a product for diagnosing or auxiliary diagnosing RARG-AML patients containing the NUP98-RARG fusion gene;

[0047] R6) Use of the primer-probe combination for detecting the PML-RARG fusion gene or the kit for detecting the PML-RARG fusion gene in detecting or assisting in detecting the RARG fusion gene for non-diagnostic purposes, or in preparing a product for detecting or assisting in detecting the RARG fusion gene, or in preparing a product for screening or assisting in screening RARG-AML patients, or in preparing a product for diagnosing or assisting in diagnosing RARG-AML patients, or in preparing a product for screening or assisting in screening RARG-AML patients containing the PML-RARG fusion gene, or in preparing a product for diagnosing or assisting in diagnosing RARG-AML patients containing the PML-RARG fusion gene;

[0048] R7) Use of the primer-probe combination for detecting the SART3-RARG fusion gene or the kit for detecting the SART3-RARG fusion gene in detecting or assisting in detecting the RARG fusion gene for non-diagnostic purposes, or in preparing a product for detecting or assisting in detecting the RARG fusion gene, or in preparing a product for screening or assisting in screening RARG-AML patients, or in preparing a product for diagnosing or assisting in diagnosing RARG-AML patients, or in preparing a product for screening or assisting in screening RARG-AML patients containing the SART3-RARG fusion gene, or in preparing a product for diagnosing or assisting in diagnosing RARG-AML patients containing the SART3-RARG fusion gene;

[0049] The present invention has the following characteristics: comprehensive coverage: the primer-probe combination for detecting the RARG fusion gene of the present invention can detect 7 types of RARG fusion genes; accurate and reliable: the primer-probe combination for detecting the RARG fusion gene of the present invention can specifically detect the RARG fusion gene and its 7 fusion types; simple, rapid and low-cost in operation. The present invention has good application prospects.

[0050] The present invention will be further described in detail below in conjunction with specific embodiments. The given embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Comparison of the detection results of the primer-probe combination of Example 1 with Sanger sequencing results for 5 RARG-AML patients.

[0052] Figure 2 Sanger sequencing results for patient 1.

[0053] Figure 3 Sanger sequencing results for patient 2.

[0054] Figure 4 Sanger sequencing results for patient 3.

[0055] Figure 5 Sanger sequencing results for patient 4.

[0056] Figure 6 Sanger sequencing results for patient 5.

[0057] Figure 7 For patient 2, the primer-probe combination of Example 1 was used to detect the mRNA level of the HNRNPC-RARG fusion gene during the initial diagnosis and follow-up during chemotherapy, and compared with the WT1 mRNA level. "Induction remission" indicates complete remission obtained by induction chemotherapy, "Consolidation 1" indicates the first consolidation course, "Consolidation 2" indicates the second consolidation course, and "Consolidation 3" indicates the third consolidation course.

[0058] Figure 8 For patient 3, the primer-probe combination of Example 1 was used to detect the mRNA level of the HNRNPC-RARG fusion gene after HSCT during follow-up, and compared with the WT1 mRNA level. Detailed implementation mode

[0059] In the following examples, the experimental methods are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified. In the following examples, for quantitative tests, at least three repeated experiments are set, and the results are averaged.

[0060] Example 1. Preparation of primer-probe combination for detecting RARG fusion gene

[0061] This example provides a primer-probe combination for detecting the RARG fusion gene, and primer-probe combinations for detecting 7 types of RARG fusion genes (CPSF6-RARG, HNRNPC-RARG, HNRNPM-RARG, NPM1-RARG, NUP98-RARG, PML-RARG, SART3-RARG).

[0062] The primer-probe combination for detecting the RARG fusion gene consists of primers 1-12 and probe 1-3, and the molar ratios of primers 1-12 to probe 1-3 are 3:3:3:3:3:3:3:3:3:3:3:3:2:2:2 in sequence.

[0063] The primer-probe combination for detecting the CPSF6-RARG fusion gene consists of primers 1-2, 10-12 and probe 1-3, and the molar ratios of primers 1-2, 10-12 and probe 1-3 are 3:3:3:3:3:2:2:2 in sequence.

[0064] The primer-probe combination for detecting the HNRNPC-RARG fusion gene consists of primers 3-4, 10-12 and probe 1-3, and the molar ratios of primers 3-4, 10-12 and probe 1-3 are 3:3:3:3:3:2:2:2 in sequence.

[0065] The primer-probe combination for detecting the HNRNPM-RARG fusion gene consists of primer 5, 10-12 and probe 1-3, and the molar ratios of primer 5, 10-12 and probe 1-3 are 3:3:3:3:2:2:2 in sequence.

[0066] The primer-probe combination for detecting the NPM1-RARG fusion gene consists of primer 6, 10-12 and probe 1-3, and the molar ratios of primer 6, 10-12 and probe 1-3 are 3:3:3:3:2:2:2 in sequence.

[0067] The primer-probe combination for detecting the NUP98-RARG fusion gene consists of primer 7, 10-12 and probe 1-3, and the molar ratios of primer 7, 10-12 and probe 1-3 are 3:3:3:3:2:2:2 in sequence.

[0068] The primer-probe combination for detecting the PML-RARG fusion gene consists of primer 8, 10-12 and probe 1-3, and the molar ratios of primer 8, 10-12 and probe 1-3 are 3:3:3:3:2:2:2 in sequence.

[0069] The primer-probe combination for detecting the SART3-RARG fusion gene consists of primer 9, 10-12 and probe 1-3, and the molar ratios of primer 9, 10-12 and probe 1-3 are 3:3:3:3:2:2:2 in sequence.

[0070] In each primer-probe combination, each primer and the probe can be independently packaged. The information of each primer is as follows:

[0071] Primer 1: 5′-Cttcatggtcagaatcctgttg-3′ (SEQ ID No.1);

[0072] Primer 2: 5′-gttcctggtggggacagatt-3′ (SEQ ID No.2);

[0073] Primer 3: 5′-gacgaagactgagcggttgt-3′ (SEQ ID No.3);

[0074] Primer 4: 5′-atgtggaggcaatcttttcg-3′ (SEQ ID No.4);

[0075] Primer 5: 5′-gaaaacatgggtcgatttgg-3′ (SEQ ID No.5);

[0076] Primer 6: 5′-ggggctttgaaataacacca-3′ (SEQ ID No.6);

[0077] Primer 7: 5′-ggatttaatactacgacagccactttg-3′ (SEQ ID No.7);

[0078] Primer 8: 5′-ccgatggcttcgacgagtt-3′ (SEQ ID No.8);

[0079] Primer 9: 5′-tcaaagtggcaatcagcaac-3′ (SEQ ID No.9);

[0080] Primer 10: 5′-cacgaagcatggcttgtaga-3′ (SEQ ID No.10);

[0081] Primer 11: 5′-gtcgggcagtctcttggat-3′ (SEQ ID No.11);

[0082] Primer 12: 5′-cagctgcggtgtgagagtc-3′ (SEQ ID No.12).

[0083] The information of each probe (FAM labeled at the 5′ end and TAMRA labeled at the 3′ end) is as follows:

[0084] Probe 1: 5′-FAM-cccgggacagtcccagccta-TAMRA-3′ (SEQ ID No.13);

[0085] Probe 2: 5′-FAM-cgagctgggcaccatctcct-TAMRA-3′ (SEQ ID No.14);

[0086] Probe 3: 5′-FAM-cctgcccactgggcctcca-TAMRA-3′ (SEQ ID No.15).

[0087] The steps for detecting whether the object to be tested contains the RARG fusion gene using the above primer-probe combination are as follows:

[0088] First, using the cDNA of the bone marrow sample of the subject to be tested as a template, RQ-PCR detections of RARG multiplex and ABL1 (internal reference) are performed separately. The RQ-PCR detection system for RARG multiplex is as follows: cDNA of the bone marrow sample of the subject to be tested, primers 1-12 (the concentration in the reaction system is 0.3 μM for each), probes 1-3 (the concentration in the reaction system is 0.2 μM for each), Universal Mastemix (ThermoFisher, catalog number: 4369016), and ddH2O is quantified to 20 μL.

[0089] The RQ-PCR detection system for ABL1 is as follows: cDNA of the bone marrow sample of the subject to be tested, primers 13-14 (the concentration in the reaction system is 0.3 μM for each), probe 4 (the concentration in the reaction system is 0.2 μM), Universal Mastemix (ThermoFisher, catalog number: 4369016), and ddH2O is quantified to 20 μL.

[0090] Primer 13: 5′-tggagataacactctaagcataactaaaggt-3′ (SEQ ID No.16);

[0091] Primer 14: 5′-gatgtagttgcttgggaccca-3′ (SEQ ID No.17);

[0092] Probe 4: 5′-FAM-ccatttttggtttgggcttcacaccatt-TAMRA-3′ (SEQ ID No.18).

[0093] If there is amplification in the reaction system of the internal reference ABL1 but no amplification in the RARG multiplex reaction system, it indicates that both are negative, that is, the subject to be tested has no RARG fusion gene and is not a RARG-AML patient; if there is amplification in the reaction system of the internal reference ABL1 and also amplification in the RARG multiplex reaction system, the subject to be tested has a RARG fusion gene and is a RARG-AML patient, and a second round of RQ-PCR detection is performed (that is, using the primer-probe combinations for detecting 7 types of RARG fusion genes to detect respectively), and the fusion gene type is determined according to the amplification results of each system; if there is no amplification in the reaction system of the internal reference ABL1, the reaction needs to be performed again.

[0094] The system for RQ-PCR detection using the primer-probe combination for detecting the CPSF6-RARG fusion gene is as follows: cDNA of the bone marrow sample of the subject to be tested, primers 1-2, 10-12 (the concentration in the reaction system is 0.3 μM for both), probe 1-3 (the concentration in the reaction system is 0.2 μM for both), universal Mastemix (ThermoFisher, catalog number: 4369016), and ddH2O is quantified to 20 μL. If there is no amplification in the reaction system for detecting the CPSF6-RARG fusion gene, the subject to be tested has no CPSF6-RARG fusion gene and is not a RARG-AML patient with the CPSF6-RARG fusion gene; if there is amplification in the reaction system for detecting the CPSF6-RARG fusion gene, the subject to be tested has the CPSF6-RARG fusion gene and is a RARG-AML patient with the CPSF6-RARG fusion gene.

[0095] The system for RQ-PCR detection using the primer-probe combination for detecting the HNRNPC-RARG fusion gene is as follows: cDNA of the bone marrow sample of the subject to be tested, primers 3-4, 10-12 (the concentration in the reaction system is 0.3 μM for both), probe 1-3 (the concentration in the reaction system is 0.2 μM for both), universal Mastemix (ThermoFisher, catalog number: 4369016), and ddH2O is quantified to 20 μL. If there is no amplification in the reaction system for detecting the HNRNPC-RARG fusion gene, the subject to be tested has no HNRNPC-RARG fusion gene and is not a RARG-AML patient with the HNRNPC-RARG fusion gene; if there is amplification in the reaction system for detecting the HNRNPC-RARG fusion gene, the subject to be tested has the HNRNPC-RARG fusion gene and is a RARG-AML patient with the HNRNPC-RARG fusion gene.

[0096] The system for RQ-PCR detection using the primer-probe combination for detecting the HNRNPM-RARG fusion gene is as follows: cDNA of the bone marrow sample of the subject to be tested, primer 5, 10-12 (the concentration in the reaction system is 0.3 μM for both), probe 1-3 (the concentration in the reaction system is 0.2 μM for both), universal Mastemix (ThermoFisher, catalog number: 4369016), and ddH2O is quantified to 20 μL. If there is no amplification in the reaction system for detecting the HNRNPM-RARG fusion gene, the subject to be tested has no HNRNPM-RARG fusion gene and is not a RARG-AML patient with the HNRNPM-RARG fusion gene; if there is amplification in the reaction system for detecting the HNRNPM-RARG fusion gene, the subject to be tested has the HNRNPM-RARG fusion gene and is a RARG-AML patient with the HNRNPM-RARG fusion gene.

[0097] The system for RQ-PCR detection using the primer-probe combination for detecting the NPM1-RARG fusion gene is as follows: cDNA of the bone marrow sample of the subject to be tested, primers 6, 10-12 (the concentration in the reaction system is 0.3 μM for each), probes 1-3 (the concentration in the reaction system is 0.2 μM for each), universal Mastemix (ThermoFisher, product number: 4369016), and ddH2O is quantified to 20 μL. If there is no amplification in the reaction system for detecting the NPM1-RARG fusion gene, the subject to be tested has no NPM1-RARG fusion gene and is not a RARG-AML patient with the NPM1-RARG fusion gene; if there is amplification in the reaction system for detecting the NPM1-RARG fusion gene, the subject to be tested has the NPM1-RARG fusion gene and is a RARG-AML patient with the NPM1-RARG fusion gene.

[0098] The system for RQ-PCR detection using the primer-probe combination for detecting the NUP98-RARG fusion gene is as follows: cDNA of the bone marrow sample of the subject to be tested, primers 7, 10-12 (the concentration in the reaction system is 0.3 μM for each), probes 1-3 (the concentration in the reaction system is 0.2 μM for each), universal Mastemix (ThermoFisher, product number: 4369016), and ddH2O is quantified to 20 μL. If there is no amplification in the reaction system for detecting the NUP98-RARG fusion gene, the subject to be tested has no NUP98-RARG fusion gene and is not a RARG-AML patient with the NUP98-RARG fusion gene; if there is amplification in the reaction system for detecting the NUP98-RARG fusion gene, the subject to be tested has the NUP98-RARG fusion gene and is a RARG-AML patient with the NUP98-RARG fusion gene.

[0099] The system for RQ-PCR detection using the primer-probe combination for detecting the PML-RARG fusion gene is as follows: cDNA of the bone marrow sample of the subject to be tested, primers 8, 10-12 (the concentration in the reaction system is 0.3 μM for each), probes 1-3 (the concentration in the reaction system is 0.2 μM for each), universal Mastemix (ThermoFisher, product number: 4369016), and ddH2O is quantified to 20 μL. If there is no amplification in the reaction system for detecting the PML-RARG fusion gene, the subject to be tested has no PML-RARG fusion gene and is not a RARG-AML patient with the PML-RARG fusion gene; if there is amplification in the reaction system for detecting the PML-RARG fusion gene, the subject to be tested has the PML-RARG fusion gene and is a RARG-AML patient with the PML-RARG fusion gene.

[0100] The system for RQ-PCR detection using a primer-probe combination for detecting the SART3-RARG fusion gene is as follows: cDNA of the bone marrow sample of the subject to be tested, primers 9, 10 - 12 (each with a concentration of 0.3 μM in the reaction system), probes 1 - 3 (each with a concentration of 0.2 μM in the reaction system), Universal Mastemix (ThermoFisher, catalog number: 4369016), and ddH2O is quantified to 20 μL. If there is no amplification in the reaction system for detecting the SART3-RARG fusion gene, the subject does not have the SART3-RARG fusion gene and is not a RARG-AML patient with the SART3-RARG fusion gene; if there is amplification in the reaction system for detecting the SART3-RARG fusion gene, the subject has the SART3-RARG fusion gene and is a RARG-AML patient with the SART3-RARG fusion gene.

[0101] Among them, no amplification: no exponential amplification curve (CT value > 40); amplification: there is an exponential amplification curve (CT value < 40).

[0102] The reaction conditions for each system are: 50 °C for 2 min, 1 cycle; 95 °C for 10 min, 1 cycle; 95 °C for 15 s, 62 °C for 1 min, 40 cycles.

[0103] Example 2. Application of the primer-probe combination of Example 1 in detecting the RARG fusion gene

[0104] I. Detecting the RARG fusion gene of RARG-AML patients using the primer-probe combination of Example 1

[0105] For 5 bone marrow samples of newly diagnosed RARG-AML patients (with patient informed consent) detected by transcriptome sequencing to have RARG rearrangement, detection is carried out using the primer-probe combination of Example 1 according to the "steps for detecting whether a subject contains the RARG fusion gene using the primer-probe combination" in Example 1. Among them, the RARG multiplex RQ-PCR system of patient 4 showed an exponential amplification curve, indicating that the patient has one of the RARG-related fusion genes. To determine the partner gene, various fusion types were detected separately. Only the detection system for the CPSF6-RARG fusion gene had an exponential amplification curve, and the others had no amplification. Therefore, it was determined that the patient has the CPSF6-RARG fusion gene.

[0106] The detection results of the fusion genes of each patient are shown in Figure 1 .

[0107] The results showed that patient 1 contains the CPSF6-RARG fusion gene and is a RARG-AML patient with the CPSF6-RARG fusion gene ( Figure 1)。The fusion of the RARG gene was detected by Sanger sequencing, and the results showed that the 4th exon of the CPSF6 gene was fused with the 2nd exon of the RARG gene ( Figure 2 ), which was consistent with the detection results using the primer-probe combination of Example 1. The primers used for Sanger sequencing were Primer 1-2 and 10-12.

[0108] The results showed that Patient 2 contained the HNRNPC-RARG fusion gene and was a patient with RARG-AML carrying the HNRNPC-RARG fusion gene ( Figure 1 )。The fusion of the RARG gene was detected by Sanger sequencing, and the results showed that the 2nd exon of the HNRNPC gene was fused with the 4th exon of the RARG gene ( Figure 3 ), which was consistent with the detection results using the primer-probe combination of Example 1. The primers used for Sanger sequencing were Primer 3-4 and 10-12.

[0109] The results showed that Patient 3 contained the HNRNPC-RARG fusion gene and was a patient with RARG-AML carrying the HNRNPC-RARG fusion gene ( Figure 1 )。The fusion of the RARG gene was detected by Sanger sequencing, and the results showed that the 3rd exon of the HNRNPC gene was fused with the 4th exon of the RARG gene ( Figure 4 ), which was consistent with the detection results using the primer-probe combination of Example 1. The primers used for Sanger sequencing were Primer 3-4 and 10-12.

[0110] The results showed that Patient 4 contained the CPSF6-RARG fusion gene and was a patient with RARG-AML carrying the CPSF6-RARG fusion gene ( Figure 1 )。The fusion of the RARG gene was detected by Sanger sequencing, and the results showed that the 4th exon of the CPSF6 gene was fused with the 4th exon of the RARG gene ( Figure 5 ), which was consistent with the detection results using the primer-probe combination of Example 1. The primers used for Sanger sequencing were Primer 1-2 and 10-12.

[0111] The results showed that Patient 5 contained the CPSF6-RARG fusion gene and was a patient with RARG-AML carrying the CPSF6-RARG fusion gene ( Figure 1 )。The fusion of the RARG gene was detected by Sanger sequencing, and the results showed that the 5′ end of the 6th exon of the CPSF6 gene was fused with the 3′ end of the 4th exon of the RARG gene ( Figure 6), which is consistent with the detection result using the primer-probe combination of Example 1. The primers used for Sanger sequencing are Primer 1-2 and 10-12.

[0112] The above experimental results show that the primer combination of Example 1 can detect various RARG fusion genes and can be used for screening RARG-AML patients.

[0113] II. Follow-up detection of RARG fusion genes in RARG-AML patients after treatment

[0114] 1. Follow-up result ①

[0115] For the above-mentioned Patient 2, the bone marrow samples at the time of initial diagnosis, after achieving complete remission in induction chemotherapy, during Consolidation Course 1, Consolidation Course 2, and Consolidation Course 3 were respectively tested for the levels of HNRNPC-RARG fusion gene and WT1 (wilms tumor 1) mRNA. The detection of the mRNA level of the HNRNPC-RARG fusion gene was carried out using the primer-probe combination for detecting the HNRNPC-RARG fusion gene in Example 1, according to the corresponding reaction system and reaction conditions in Example 1. The primers and probe used for detecting WT1 are as follows: Forward primer: 5′-GATAACCACACAACGCCCATC-3′; Reverse primer: 5′

[0116] -CACACGTCGCACATCCTGAAT-3′; Probe: 5′-FAM-ACACCGTGCGTGTGTATTCTGTATTGG-TAMRA-3′.

[0117] The results are as Figure 7 shown. During the process of consolidation treatment, the two showed good consistency when WT1 was abnormally expressed. The mRNA level of the HNRNPC-RARG fusion gene gradually decreased, indicating that the primer combination of Example 1 can be used to detect MRD in RARG-AML patients. And the mRNA of the HNRNPC-RARG fusion gene could still be detected when WT1 was normal (<0.6%), indicating that the primer-probe combination of Example 1 has high sensitivity.

[0118] 2. Follow-up result ②

[0119] The above-mentioned Patient 3 received allogeneic hematopoietic stem cell transplantation (HSCT) after induction remission consolidation chemotherapy. The bone marrow samples after transplantation were respectively tested for the levels of HNRNPC-RARG fusion gene and WT1 (wilms tumor 1) mRNA. The detection of the mRNA level of the HNRNPC-RARG fusion gene was carried out using the primer-probe combination for detecting the HNRNPC-RARG fusion gene in Example 1, according to the corresponding reaction system and reaction conditions in Example 1.

[0120] The results showed that there was an abnormal increase in the WT1 mRNA level at HSCT + 44 months (5.6%, and the normal value of the WT1 mRNA level was <0.6%). Figure 8 The levels of the HNRNPC-RARG fusion gene and WT1 mRNA at each time point since HSCT + 44 months were shown. The two showed good consistency when WT1 was abnormally expressed, and the mRNA of the HNRNPC-RARG fusion gene could still be detected when WT1 was normal (HSCT + 48 months), indicating that the primer-probe combination of Example 1 had high sensitivity.

[0121] III. Screening of negative samples

[0122] Samples to be tested: Bone marrow samples of 20 non-RARG-AML patients (10 were patients with acute promyelocytic leukemia positive for PML-RARA, and 10 were patients with other types of acute myeloid leukemia in complete remission).

[0123] The above samples to be tested were detected using the primer-probe combination for detecting the RARG fusion gene in Example 1, according to the corresponding reaction system and reaction conditions in Example 1. The results showed that all 20 samples to be tested were negative, indicating that the primer-probe combination for detecting the RARG fusion gene in Example 1 had high specificity.

[0124] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that are outside the scope disclosed in this application.

Claims

1. A primer-probe combination for detecting RARG fusion gene, comprising primers 1-12 and probes 1-3, wherein the primers 1-12 are 12 single-stranded DNAs shown in SEQ ID No. 1-12 in the sequence list, and the sequences of the probes 1-3 are SEQ ID No. 13, 14 and 15 in the sequence list; Both ends of the probes 1-3 are labeled with a fluorescent group and a fluorescent quenching group, respectively.

2. A kit for detecting RARG fusion gene, comprising the primer-probe combination according to claim 1.

3. Any of the following uses of the primer-probe combination according to claim 1 or the kit according to claim 2: X1) preparing products for detecting or assisting in detecting RARG fusion genes, wherein the RARG fusion genes are CPSF6-RARG and / or HNRNPC-RARG; X2) preparing products for screening or assisting in the screening of patients with RARG-AML; X3) Preparation of products for screening or assisting in screening of non-RARG-AML patients; X4) preparing products for diagnosis or aiding diagnosis of RARG-AML patients; The RARG-AML patients are RARG-AML patients caused by CPSF6-RARG and / or HNRNPC-RARG.

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