MYB gene transcript detection primer combination, kit and method for evaluating leukemia prognosis and application of MYB gene transcript detection primer combination, kit and method

Abstract

The invention discloses an MYB gene transcript detection primer combination for evaluating leukemia prognosis, a kit, a method and application of the MYB gene transcript detection primer combination. The primer combination comprises a first primer pair for specifically detecting an MYB TSS1 transcript and a second primer pair for detecting an MYB TSS2 transcript, and the nucleotide sequences of the first primer pair and the second primer pair are respectively shown as SEQ ID NO: 1-4. The amplification efficiency of the two pairs of primers is highly consistent. The kit containing the primer combination is simple and convenient to operate and suitable for clinical conventional use. The invention further provides a method for detecting the ratio of MYB TSS2 transcripts to TSS1 transcripts in a leukemia sample through real-time fluorescent quantitative PCR by using the primer or the kit, and evaluating the prognosis of a patient according to the ratio of the MYB TSS2 transcripts to the TSS1 transcripts. The ratio can be used as a novel molecular marker for predicting the refractory, drug resistance and recurrence risk of leukemia patients (especially myelogenous and gonorrhea leukemia). The invention provides an effective tool for precise prognosis stratification of leukemia.

Description

Technical Field

[0001] This invention belongs to the field of tumor molecular diagnostics and in vitro diagnostic reagents technology, specifically relating to a molecular detection tool and method for assessing the prognosis of leukemia, particularly to specific primers for detecting transcripts at different transcription start sites (TSS) of the MYB gene, a kit containing the primers, a method for detection and risk assessment using the kit, and its use in the preparation of leukemia prognosis assessment products. Background Technology

[0002] Despite advancements in risk stratification, intensive chemotherapy, hematopoietic stem cell transplantation, and targeted / immunotherapy in recent years, refractory, relapse, and drug resistance remain the main factors affecting long-term survival in leukemia. Current prognostic assessments for leukemia primarily rely on the following aspects: first, cytogenetic and molecular genetic abnormalities, such as fusion genes, chromosome number and structural abnormalities, and gene mutation profiles; second, minimal residual disease (MRD) detection, including methods based on flow cytometry, qPCR / NGS for fusion genes, or clonal rearrangements; and third, clinical risk factors, such as age, white blood cell count, and initial treatment response. However, existing biomarkers and risk stratification systems still have significant limitations. Many patients exhibit poor or no remission (refractory) after initial induction therapy, but simple, stable molecular markers are lacking for further prognostic subdivision of this population. Some patients classified as intermediate-risk or relatively low-risk in traditional stratification still relapse or exhibit significant chemotherapy resistance. Most existing molecular markers rely on specific structural alterations (such as specific fusion genes or mutations) and are not applicable to all types of leukemia. In the initial diagnosis stage, clinicians often lack reliable molecular indicators to predict the risk of subsequent refractory, drug-resistant and relapse, which is not conducive to the early development of individualized intensive treatment strategies.

[0003] MYB is an important transcription factor, highly expressed in leukemia, regulating the proliferation, differentiation, and invasion of cancer cells, and is a driving factor in leukemia development. Abnormal MYB activation has been shown to promote leukemia occurrence, progression, and drug resistance, and is essential for maintaining the proliferation of primary human leukemia cells. Recent studies have revealed different transcription start sites for the MYB gene. The classical MYB promoter (TSS1), located upstream of exon 1, can respond to various stimuli to produce the full-length MYB. Furthermore, a selective MYB promoter (TSS2), located upstream of exon 2, is associated with aberrant MYB expression in certain leukemia cell lines; it expresses a truncated MYB variant lacking the first 20 amino acids. In acute lymphoblastic leukemia (ALL), existing literature has used RNA-Seq and qPCR to analyze transcripts of different MYB promoters, suggesting that MYB TSS2 activity is associated with relapse and drug resistance.

[0004] Nevertheless, the existing technology has significant shortcomings: First, the disease scope is relatively limited, with previous reports mainly focusing on childhood ALL, and a unified detection protocol that can be widely applied has not yet been established; second, the detection system is not yet standardized, with existing studies based on experimental qPCR discovered through RNA-seq, lacking a set of rigorously validated standardized primer combinations and kits suitable for routine clinical testing; third, there is a lack of specific molecular prognostic biomarkers for patients with unresolved or refractory leukemia. Therefore, it is necessary to develop a standardized qPCR detection primer, kit, and supporting detection method based on different MYB transcript ratios that can be used in myeloid and lymphoid leukemia for prognostic assessment of leukemia patients (including newly diagnosed, unresolved / refractory, and relapsed stages), predicting the risk of subsequent refractory, chemotherapy resistance, and relapse. Summary of the Invention

[0005] To address the aforementioned deficiencies in existing technologies, this invention provides primers, reagent kits, and protocols for routine clinical testing that are simple to operate, cost-effective, and suitable for accurately assessing the prognostic risk of patients with myeloid and lymphoid leukemia. This invention creatively uses the MYB TSS2 / TSS1 transcript ratio as a prognostic biomarker for leukemia (including myeloid and lymphoid), overcoming the shortcomings of existing prognostic stratification systems, and particularly providing a new molecular tool for identifying patients with refractory / non-remission and high relapse risk. The two pairs of specific primers provided in this invention have undergone rigorous validation, exhibiting high and closely approximate amplification efficiencies, ensuring the accuracy and comparability of the ratio calculation, and overcoming the low standardization of research qPCR methods.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides a set of specific primer combinations for detecting MYB gene transcripts, comprising: a first primer pair (MYB TSS1-F / R) for detecting MYB TSS1 transcripts and / or a second primer pair (MYB TSS2-F / R) for detecting MYB TSS2 transcripts, wherein: The first primer pair includes: The upstream primer MYB TSS1-F has a preferred nucleotide sequence as shown in SEQ ID NO:1; The downstream primer MYB TSS1-R has a preferred nucleotide sequence as shown in SEQ ID NO:2.

[0007] The second primer pair includes: The upstream primer MYB TSS2-F has a preferred nucleotide sequence as shown in SEQ ID NO:3; The downstream primer MYB TSS2-R has a preferred nucleotide sequence as shown in SEQ ID NO:4.

[0008] MYB TSS1-F / R and MYB TSS2-F / R are two specific primer pairs used to detect transcripts from different transcription start sites of the MYB gene.

[0009] Experimental verification showed that the primer pairs used to detect MYB TSS1 transcripts and those used to detect MYB TSS2 transcripts had essentially the same amplification efficiency, exhibiting high specificity for their respective target transcripts. Furthermore, their amplification efficiencies were similar (e.g., both within the 90%-110% range, with a difference of less than 5%), and the standard curves showed excellent linearity (R²>0.999). This provides a reliable methodological foundation for the subsequent accurate calculation of the TSS2 / TSS1 transcript ratio using the 2^-ΔCt method. In one specific embodiment, under the same real-time quantitative PCR reaction system and cycling conditions, the slope of the standard curve for the primer pair used to detect MYB TSS1 was determined by the standard curve method. The correlation coefficient R² = 0.9975, corresponding to an amplification efficiency of approximately 91.2%, is 3.553. The slope of the standard curve for the primer pair used to detect MYB TSS2 is... The correlation coefficient R² = 0.9990 corresponds to an amplification efficiency of approximately 90.8%. The amplification efficiencies of the two primer pairs are highly similar, which helps ensure the accuracy and comparability of the MYB TSS2 / TSS1 transcript ratio calculated based on the 2^-ΔCt method.

[0010] A second aspect of the present invention provides a diagnostic kit for leukemia prognostic assessment comprising the above-described primer combination, the kit comprising at least: MYB TSS1 specific primer pair (MYB TSS1-F / R) and MYB TSS2 specific primer pair (MYB TSS2-F / R); the primer pairs are preferably provided in lyophilized powder or premixed form.

[0011] Preferably, the kit may also contain one or more of the following components: a) real-time quantitative PCR reaction solution; b) a positive control, which is a standard plasmid containing the corresponding target sequences of MYB TSS1 and TSS2, used to verify the performance of the reaction system and primers; c) a negative control; d) quantitative standards or serially diluted standards for establishing a standard curve.

[0012] The real-time quantitative PCR reaction solution includes DNA polymerase, dNTPs, and Mg²⁺. +Components such as buffer solution can be in the form of 2× or 5× premixed solution; The negative control was deionized water, used to monitor for contamination and nonspecific amplification. Quantitative standards or serially diluted standards are used for absolute or relative quantitative analysis; Since the kit described in this invention uses qRT-PCR technology for detection, the commonly used reagents in the kit can be purchased separately from the market or prepared by the customer. Therefore, the specific reagents to be included in the kit can be configured according to the customer's actual needs. For convenience, all reagents can also be included in the kit.

[0013] Preferably, the kit is designed as a commercial in vitro diagnostic kit that can be used directly on a conventional real-time quantitative PCR instrument, making it suitable for routine use in hospital laboratories and third-party laboratories.

[0014] A third aspect of the present invention provides a method for determining the MYBTSS2 / TSS1 transcript ratio in leukemia samples. The method includes the following steps: (1) Sample processing: Obtaining total RNA from the leukemia sample to be tested (such as bone marrow or peripheral blood samples, extracted using conventional methods) and reverse transcribing it into cDNA. (2) Real-time quantitative PCR: Using the cDNA obtained in step (1) as a template, performing qPCR reactions using the first primer pair and the second primer pair described above, respectively, to obtain their respective Ct values. (3) Ratio calculation: Calculating the relative expression ratio of MYB TSS2 transcripts to MYB TSS1 transcripts based on the Ct values ​​obtained in step (2). The obtained ratio can be used to assess the prognostic risk of patients.

[0015] A fourth aspect of the invention provides the use of the above-described specific primer combination and the detection kit comprising it in the preparation of products for prognostic assessment or risk prediction of leukemia. The products can be used to assess the risk of refractory, chemotherapy-resistant, and relapsed leukemia patients (including but not limited to acute myeloid leukemia, acute lymphoblastic leukemia, etc.).

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) Wide applicability Using the MYB TSS2 / TSS1 transcript ratio as the core indicator, combined with a validated specific primer combination and a standardized qPCR system, this method can be applied to various subtypes of myeloid and lymphoid leukemia, and has broad-spectrum prognostic assessment capabilities.

[0017] (2) Prognostic assessment for patients with non-remission / refractory disease Through a systematic study of patients with unremission / refractory leukemia, this invention demonstrates that the TSS2 / TSS1 ratio can serve as an independent prognostic factor for this population, guiding subsequent intensive treatment or transplantation decisions.

[0018] (3) Prognostic assessment is moved to the initial diagnosis stage. This invention detects the MYB TSS2 / TSS1 transcript ratio in newly diagnosed leukemia patients and finds that those with an elevated ratio have a significantly increased proportion of refractory / non-remission, chemotherapy resistance, and relapse during follow-up. This allows for stratification of subsequent treatment response and relapse risk before treatment begins, which is beneficial for developing individualized treatment and follow-up strategies at an early stage.

[0019] (4) The test is simple and can be promoted clinically. The real-time quantitative PCR platform is simple to operate, the equipment is universal, and it is suitable for hospitals and testing institutions at all levels. The amplified fragments are short, the requirements for sample RNA quality are relatively low, and it is compatible with some degraded samples.

[0020] (5) Standardizable reagent kit format Providing specific primers, positive / negative controls, and standard operating procedures in the form of a kit facilitates mass production and quality control, and improves the comparability and reproducibility of test results between different laboratories.

[0021] (6) It can complement the existing hierarchical system. The indicators of this invention can be combined with existing indicators such as cytogenetics, gene mutation, and MRD to further refine risk stratification, improve the accuracy of prognostic assessment, and provide new molecular indicators for the joint stratification of "biology + response". Attached Figure Description

[0022] Figure 1 This is a graph showing the PCR efficiency analysis of MYB TSS2 and TSS1; Figure 2 This is a distribution map of the MYB TSS2 / TSS1 ratio in the bone marrow of leukemia patients with different treatment outcomes. Detailed Implementation

[0023] The following embodiments further clearly and completely describe the content of the present invention. These embodiments are merely a part of the present invention and should not be construed as limiting the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without inventive effort are within the scope of protection of the present invention.

[0024] Example 1: Performance Verification of MYB TSS1 / TSS2 Specific Primers Plasmids containing complete MYB cDNA fragments were selected as templates and serially diluted 10-fold (10... 6 ~10¹ copies / reaction), using the TSS1-specific primer pair and TSS2-specific primer pair of the present invention for real-time quantitative PCR amplification. The Ct values ​​of each gradient were plotted against the logarithmic template concentration, and a standard curve was obtained using linear regression. The PCR efficiency was calculated as (10^(-1 / slope). 1) Calculate the amplification efficiency by multiplying by 100%.

[0025] The results showed that the standard curve equation for the primer pair used to detect MYB TSS1 was y = 3.553x + 28.14, R² = 0.9975, corresponding to an amplification efficiency of approximately 91.2%; the standard curve equation for the primer pair used to detect MYB TSS2 is y = 3.564x + 28.26, R² = 0.9990, corresponding to an amplification efficiency of approximately 90.8%. The amplification efficiencies of both primer pairs are within the range of 90%–110%, with a difference of less than 5%, indicating that the TSS1 and TSS2 specific primers of this invention have essentially the same amplification efficiency under the same reaction conditions, making them suitable for reliable calculation of the MYB TSS2 / TSS1 transcript ratio using the 2^-ΔCt method.

[0026] Example 2: Composition and preparation of the reagent kit This embodiment provides a real-time quantitative PCR kit for detecting the MYB TSS2 / TSS1 transcript ratio in leukemia patients. The kit composition is as follows (the formulation and specifications of each component are only preferred embodiments, and those skilled in the art can make appropriate adjustments without departing from the spirit of the invention).

[0027] (1) MYB TSS1 specific primer pair (component A) Upstream primer MYB TSS1-F (SEQ ID NO:1) Downstream primer MYB TSS1-R (SEQ ID NO:2) Stock solution concentration: 10 μmol / L each. The final concentration after adding to the reaction system according to the instructions is 0.2 μmol / L. It can be dispensed into 0.5 mL tubes; each tube is sufficient for approximately 100 reactions.

[0028] (2) MYB TSS2 specific primer pair (component B) Upstream primer MYB TSS2-F (SEQ ID NO:3) Downstream primer MYB TSS2-R (SEQ ID NO:4) Stock solution concentration: 10 μmol / L each, final concentration at use: 0.2 μmol / L.

[0029] (3) 2× Real-time Quantitative PCR Premix (Component C) Contains: hot-start DNA polymerase; dNTP mixture; Mg²⁺ + Suitable buffer solution; fluorescent dye (such as SYBR Green or equivalent dye).

[0030] When using, add at a concentration of 2×, adding 10 μL to each reaction tube.

[0031] (4) Positive control (component D) It contains a standard template that expresses the full length of MYB, preferably a plasmid mixture or standard cDNA containing the corresponding cDNA fragment.

[0032] After quantification, it is prepared as a working solution and can be directly used for qPCR reactions to verify primer and system performance.

[0033] (5) Negative control (component E) Template-free controls (nuclease-free water or buffer) are used to monitor for contamination and nonspecific amplification.

[0034] (6) Optional standard (component F) If absolute or relative quantification is required, 3 to 5 standards with different concentration gradients can be provided to establish a standard curve.

[0035] (7) Instructions for use (Component G) It is stated that: Applicable sample types (bone marrow / peripheral blood mononuclear cells, etc.); Recommended RNA extraction and reverse transcription methods; Recommended PCR reaction system ratio and cycling conditions (e.g., 95℃ pre-denaturation for 3 min, followed by 40 cycles: 95℃ for 15 s, 60℃ for 30 s). Results analysis methods, including ΔCt, 2^-ΔCt calculations, and the formula for calculating the MYB TSS2 / TSS1 ratio; reference threshold ranges obtained in the acute leukemia cohort for prognostic risk stratification.

[0036] Storage conditions: Primers and positive controls are preferred. Store at 20℃; 2× qPCR premixed solution at 2–8℃ or Store at 20℃; the shelf life of the reagent kit is preferably 12 months from the date of manufacture.

[0037] The formulation and packaging described in this embodiment allow for the direct detection of MYB TSS1 and MYB TSS2 on a conventional real-time quantitative PCR platform, and the calculation of the MYB TSS2 / TSS1 transcript ratio. This can be used for prognostic assessment of leukemia patients and prediction of the risk of non-remission / refractory / drug-resistant / relapse.

[0038] Example 3: Method for detecting the MYB TSS2 / TSS1 ratio using a kit The kit in this embodiment is the same as the kit provided in Example 2. The detection steps include total RNA extraction, reverse transcription reaction, and real-time quantitative PCR detection. Specific steps are as follows: (1) Total RNA extraction Total RNA was extracted using the Trizol method. 1 mL of Trizol reagent was added to the sample, homogenized, and lysed for 5 min.

[0039] Add 200 μL of chloroform, mix the solution thoroughly, and let it stand at room temperature for 3 min; Pre-cooled centrifuge, 4°C, 12,000 rpm, centrifuge for 15 min; Transfer the upper aqueous phase to another 1.5 mL EP tube, add 0.5 mL isopropanol, and incubate for 10 min; Centrifuge at 4°C, 12,000 rpm for 10 min. Discard the supernatant. Add 1 mL of 75% ethanol (prepared with DEPC in water) and vortex to suspend the sample. Centrifuge at 7500 g for 5 min at 4°C. Repeat washing twice. Discard the ethanol, collect the RNA (white substance), and air-dry the RNA in the EP tube for 10 min. Add 50 µL of DEPC water, incubate at 55°C for 10 min to completely dissolve the RNA, and then freeze at -80°C.

[0040] (2) Reverse transcription reaction The reagents used included random primers, cDNA strand synthesis buffer, genomic removal reagent, genomic removal buffer, cDNA strand synthase mixture, cDNA strand synthesis buffer, and RNase-free water, purchased from TakaRa (#RR047A).

[0041] Genomic DNA removal reaction: Prepare the reaction mixture on ice according to the table below, place the PCR tube containing the reaction solution in the PCR instrument, incubate at 42°C for 2 min, and then store at 4°C; Reverse transcription reaction: Add the ingredients listed in the table below to the PCR tube according to the corresponding usage amount to prepare the reaction solution. Perform the experiment on ice. After the reaction system is prepared, mix it well and centrifuge briefly to collect the solution on the tube wall to the bottom of the tube.

[0042] cDNA synthesis reaction conditions: 37°C for 15 min, 85°C for 5 s, and store at 4°C.

[0043] (3) RT-qPCR reaction The reagents used for real-time quantitative PCR included 2×qRT-PCR amplification enzyme mixture and deionized water, purchased from TransGen Biotech (AQ101-01). The following reaction system was prepared in a PCR tube and thoroughly mixed by gently pipetting. Each sample required independent reaction using MYB TSS1 and MYB TSS2 primer pairs.

[0044] The PCR reaction program was as follows: 95°C, 30 s pre-denaturation; 95°C, 5 s denaturation; 60°C, 30 s amplification; for a total of 35 cycles. The Ct values ​​of MYB TSS1 and MYB TSS2 for each sample were recorded (average of replicate wells), and the MYB TSS2 / TSS1 transcript ratio was calculated using the 2^-ΔCt method.

[0045] Example 4: Detection of the MYB TSS2 / TSS1 transcript ratio in bone marrow samples from leukemia patients and its relationship with prognosis. This embodiment uses the kit described in Example 2 and the detection method described in Example 3 to detect the MYB TSS2 / TSS1 transcript ratio in bone marrow samples from patients with acute leukemia, and analyzes its relationship with clinical outcomes such as remission, non-remission, refractory, and relapse.

[0046] (1) Research subjects and grouping Based on treatment response and follow-up outcomes, patients were divided into: Remission group: Those who achieved and maintained complete remission after induction therapy; No remission group: Those who have not achieved complete remission after the prescribed course of induction therapy; Refractory group: Patients who do not respond well to standard induction or multi-line regimens and require frequent changes in treatment regimens; Relapse group: Patients who achieved remission but experienced hematologic or myeloid relapse during follow-up.

[0047] (2) Specimen collection and processing 1–2 mL of bone marrow was collected from each patient, anticoagulated with an anticoagulant, stored at 4 °C for a short period, and subsequent processing was completed within 24 hours.

[0048] Bone marrow mononuclear cells were isolated using density gradient centrifugation. Total RNA was extracted using the Trizol method described in Example 3. After confirming the purity by measuring the A260 / A280 ratio using a spectrophotometer, reverse transcription was performed using a commercial reverse transcription kit to prepare a cDNA template. The specific RNA extraction and reverse transcription steps were the same as in Example 3 and will not be repeated here.

[0049] (3) Detection of MYB TSS2 / TSS1 transcript ratio Using the kit described in Example 2, the expression of MYB TSS1 and MYBTSS2 was detected on a real-time quantitative PCR instrument. At least three technical replicates were set for each sample. The reaction system and amplification procedure were the same as in Example 3. Ct_TSS1 and Ct_TSS2 values ​​for each sample were recorded, and the MYB TSS2 / TSS1 transcript ratio was calculated using the 2^-ΔCt method.

[0050] (4) Outcomes in AML patients In AML patients, the MYB TSS2 / TSS1 ratio was generally low in the remission group, with the vast majority of cases ranging from approximately 1 to 2. However, the ratio was significantly higher in the non-remission, refractory, and relapsed groups, with the scatter plot showing a clear upward trend and the median value being higher than that in the remission group. These results suggest that the MYB TSS2 / TSS1 ratio is also an effective predictor of poor prognosis in ALL patients.

[0051] Statistical analysis showed that the MYB TSS2 / TSS1 ratio was significantly higher in the non-remission group compared to the remission group; the difference was even more significant in the MYB TSS2 / TSS1 ratio compared to the remission group; and there was also a significant difference between the relapsed group and the remission group. These results suggest that in AML, a higher MYB TSS2 / TSS1 ratio is associated with a greater risk of adverse outcomes such as induced non-remission, refractory disease, or relapse.

[0052] (5) Outcomes in ALL patients A similar trend was observed in ALL patients. The MYB TSS2 / TSS1 ratio was lowest in the remission group, mostly concentrated in the lower range; the ratio was generally higher in the non-remission group, significantly different from the remission group; the ratio was further increased in the refractory and relapse groups, significantly higher than the remission group, with a significantly upward shift in the median. These results indicate that in ALL, an elevated MYB TSS2 / TSS1 ratio is also closely associated with refractory and relapse, and can serve as a molecular marker for assessing prognostic risk in ALL patients. A trend consistent with AML was observed. The ratio was lowest in the remission group, with the ratios increasing significantly in the non-remission, refractory, and relapse groups, and the differences between groups were statistically significant. These results suggest that the MYB TSS2 / TSS1 ratio is also an effective predictor of poor prognosis in ALL patients.

[0053] (4) Conclusion This embodiment demonstrates that, in both AML and ALL, the MYB TSS2 / TSS1 transcript ratio can effectively distinguish between patients with good prognosis (remission) and those with poor prognosis (no remission, refractory, relapse). A higher ratio indicates a greater risk of treatment ineffectiveness, drug resistance, or relapse. Combined with the highly efficient and consistent primer performance verified in Example 1, and the standardized kits and procedures provided in Examples 2 and 3, this invention establishes a complete and reliable prognostic risk assessment scheme for leukemia, possessing significant clinical value.

[0054] This embodiment demonstrates that, among patients with both AML and ALL acute leukemia, patients in remission have the lowest MYB TSS2 / TSS1 transcript ratio, while the ratio is significantly higher in patients who are not in remission, refractory, or relapsed. The MYB TSS2 / TSS1 ratio can distinguish between patients with good prognosis (remission) and those with poor prognosis (not in remission, refractory, or relapsed), and can be used for prognostic assessment and risk stratification of refractory / relapse in acute leukemia patients. Combined with the primer performance, kit composition, and operating procedures shown in Examples 1-3, the detection scheme provided by this invention has good feasibility and practical value in clinical samples.

[0055] sequence list MYB TSS1-F (SEQ ID NO:1): 5'-CGAAGACCCCGGCACAG-3' MYB TSS1-R (SEQ ID NO:2): 5'-GACGCTTTCCAGACTTGGGA-3' MYB TSS2-F (SEQ ID NO:3): 5'-ATGATGGGCTGCTTCCCAAG-3' MYB TSS2-R (SEQ ID NO:4): 5'-TCCATTCTGTTCCACCAGCTT-3'。

Claims

1. A MYB gene transcript detection primer combination for leukemia prognosis evaluation, characterized by, Comprising: a first primer pair MYB TSS1-F / R for detecting MYB TSS1 transcript, and / or a second primer pair MYB TSS2-F / R for detecting MYB TSS2 transcript, wherein: the first primer pair comprises: an upstream primer MYB TSS1-F, the nucleotide sequence of which is shown as SEQ ID NO: 1; a downstream primer MYB TSS1-R, the nucleotide sequence of which is shown as SEQ ID NO:

2. the second primer pair comprises: an upstream primer MYB TSS2-F, the nucleotide sequence of which is shown as SEQ ID NO: 3; a downstream primer MYB TSS2-R, the nucleotide sequence of which is shown as SEQ ID NO:

4.

2. A test kit for leukemia prognosis evaluation, characterized by, Comprising: a first primer pair MYB TSS1-F / R for detecting MYB TSS1 transcript, and a second primer pair MYB TSS2-F / R for detecting MYB TSS2 transcript.

3. The test kit according to claim 2, characterized in that Further comprising one or more of the following components: a) a real-time fluorescent quantitative PCR reaction solution; b) a positive control, which is a standard plasmid containing the corresponding target sequences of MYB TSS1 and TSS2; c) a negative control; d) a quantitative standard or gradient dilution standard for establishing a standard curve.

4. The test kit according to claim 3, characterized in that The primer pairs are provided in the form of lyophilized powder or premix solution.

5. The test kit according to claim 3, characterized in that, The real-time fluorescent quantitative PCR reaction solution is a 2x or 5x premix solution containing a DNA polymerase, dNTP, Mg 2+ and a buffer. The real-time fluorescent quantitative PCR reaction solution is a 2x or 5x premix solution containing a DNA polymerase, dNTP, Mg 2+ and a buffer.

6. A method of determining the MYB TSS2 / TSS1 transcript ratio in a leukemia sample, characterized in that, Comprising the following steps: (1) sample processing: obtaining total RNA of the sample to be tested and reverse transcribing it into cDNA; (2) real-time fluorescent quantitative PCR: using the cDNA obtained in step (1) as a template, performing qPCR reaction with the first primer pair and the second primer pair respectively to obtain the respective Ct values; (3) ratio calculation: calculating the relative expression ratio of MYB TSS2 transcript to MYB TSS1 transcript according to the Ct values obtained in step (2).

7. Use of the primer combination of claim 1, or the detection reagent kit of any one of claims 2-5 in the preparation of a product for the prognosis evaluation or risk prediction of leukemia.

8. Use according to claim 7, characterized in that, The product can be used to evaluate the risk of refractory, chemotherapy-resistant and relapse of leukemia patients.

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