NPM1 mutation dynamic monitoring method based on microdroplet digital PCR technology

Abstract

The invention discloses an NPM1 mutation dynamic monitoring method based on a microdroplet digital PCR technology, and relates to the technical field of molecular diagnos.The method comprises the steps that S1, RNA is extracted from a sample; s2, carrying out reverse transcription on the extracted RNA to obtain cDNA (complementary deoxyribonucleic acid); s3, designing a specific primer and a fluorescent probe aiming at the NPM1 gene exon 12 A type mutation, and designing a primer and a fluorescent probe of an ABL1 gene at the same time; s4, verifying the designed primer and probe and confirming the performance of a reaction system; s5, preparing a reaction mixed solution; s6, carrying out oil drop generation on the reaction mixed solution; specific primers and a fluorescent probe are designed for NPM1 gene exon 12 type A mutation, ABL1 is used as a reference gene, a droplet digital PCR platform is used for detection, the amplification efficiency and specificity of the primers and the probe are verified through real-time fluorescent quantitative PCR, the concentration combination of the primers and the probe is optimized by using a clinical sample, and the kit is used for detecting the NPM1 gene exon 12 type A mutation. And a plurality of samples are adopted for cross validation, so that high sensitivity and specificity of the method on NPM1 mutation detection are ensured.

Description

Technical Field

[0001] This invention relates to the field of molecular diagnostics technology, specifically to a method for dynamic monitoring of NPM1 mutations based on droplet digital PCR technology. Background Technology

[0002] The nucleophosphoprotein 1 (NPM1) gene is located on human chromosome 5q35 and contains 12 exons. The encoded NPM protein participates in a variety of key cellular physiological processes. In acute myeloid leukemia (AML), somatic mutations of the NPM1 gene are common, mainly concentrated in exon 12. These mutations are mostly four-base insertions leading to frameshift mutations, altering the nuclear output signal of the protein. NPM1 mutations are highly prevalent in AML patients and are a unique molecular marker of AML. Due to the stability of the mutation site and the well-defined sequence, it has become an important molecular target for the detection of minimal residual disease (MRD). The detection results are of great significance for the evaluation of treatment efficacy, prognosis, and treatment decisions in AML patients.

[0003] Currently, while existing NPM1 mutation detection technologies have promoted the development of this field to some extent, they all have limitations and cannot meet the high requirements of clinical MRD monitoring. PCR amplification and Sanger sequencing, as traditional detection methods, have limited sensitivity and may not accurately detect low-abundance NPM1 mutations, easily leading to false negatives and affecting the accurate assessment of patient conditions. Reverse transcription polymerase chain reaction (RT-PCR) performs poorly in terms of repeatability, and results from different batches may fluctuate significantly, making it difficult to guarantee the stability and reliability of the test results. High-throughput nucleic acid sequencing technology, while providing rich information, has a complex process involving multiple cumbersome steps such as sample preparation, library construction, sequencing, and data analysis, requiring extremely high levels of technical skill from operators and advanced laboratory equipment. The high cost limits its large-scale clinical application. Multiplex PCR combined with capillary electrophoresis also suffers from insufficient sensitivity, and may not be able to effectively identify a small number of mutant cells in minimal residual lesions. At the same time, this technology has strict requirements for experimental conditions, and any slight change in experimental conditions may affect the test results, resulting in poor reproducibility. Although digital PCR technology has improved the sensitivity and accuracy of detection to some extent, some existing digital PCR methods still have shortcomings in terms of reaction system optimization and primer and probe design, which affect the specificity and stability of the detection and cannot meet the high-precision requirements of clinical dynamic monitoring of NPM1 mutations. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a dynamic monitoring method for NPM1 mutations based on droplet digital PCR technology. This method extracts RNA from samples and reverse transcribes it into cDNA, providing a suitable template for subsequent detection. Specific primers and fluorescent probes are carefully designed for the type A mutation in exon 12 of the NPM1 gene. Simultaneously, corresponding primers and probes are designed using ABL1 as an internal reference gene. Through rigorous verification of primers and probes and confirmation of reaction system performance, the combination of primer and probe concentrations is optimized to determine the best reaction conditions. Cross-validation with multiple samples ensures the reliability of the detection results. After preparing the reaction mixture, microfluidic technology is used to divide the reaction system into a large number of uniform droplets, achieving absolute quantification of nucleic acid molecules. The template in the droplets is amplified using a specific PCR amplification program, and the fluorescence signal of the amplification product is collected using a droplet reader. Finally, the fluorescence signal data is analyzed in depth using supporting analysis software to calculate the proportion of positive droplets and convert it into the absolute copy number of the target nucleic acid. Accurate interpretation is performed by combining threshold and positive / negative control results, outputting quantitative detection results.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for dynamic monitoring of NPM1 mutations based on droplet digital PCR technology, the method comprising the following specific steps: S1. Extract RNA from the sample; S2. The extracted RNA is reverse transcribed into cDNA; S3. Design specific primers and fluorescent probes for type A mutation in exon 12 of the NPM1 gene, and design primers and fluorescent probes for the ABL1 gene. S4. Verify the designed primers and probes and confirm the performance of the reaction system; S5. Prepare the reaction mixture; S6. Generate oil droplets from the reaction mixture; S7. Perform PCR amplification on the system after oil droplets are generated; S8. Detect PCR amplification products using a droplet reader; S9. Analyze the test data and interpret the results.

[0006] Furthermore, in step S3, highly specific amplification primers and fluorescent probes are designed for the type A mutation in exon 12 of the NPM1 gene, wherein the forward primer is located in exon 10, the reverse primer spans the mutation insertion region, and the probe covers the connection region between exons 10 and 11.

[0007] Furthermore, in step S3, ABL1 is set as an internal reference gene, with its forward primer spanning exons 2 and 3, its reverse primer located in exon 3, and the probe located between the two primers.

[0008] Furthermore, in step S4, the amplification efficiency and specificity of NPM1 type A mutation and ABL1 primers and probes are verified by real-time quantitative PCR. Then, clinical bone marrow and peripheral blood samples, including positive and negative samples, are used to optimize the primer and probe concentration combination and evaluate its impact on amplification efficiency, background signal and system stability. The optimal ddPCR reaction conditions are determined, and cross-validation is further carried out using NPM1 RNA standards, HL60 cell lines and clinical samples to confirm the performance of the system.

[0009] Furthermore, in step S4, the optimized concentration of specific primers and probes, ddPCR-specific buffer, and nuclease-free water are mixed in a predetermined ratio to prepare a reaction mixture with a total volume of 20 μL. The components and amounts are as follows: Droplet PCR Supermix for Probe 10 μL, NPM1-A primer mixture 2 μL, NPM1-A-Probe 0.5 μL, ABL1 primer mixture 2 μL, ABL1-Probe 0.5 μL, template cDNA 25-250 ng, and nuclease-free water to a final volume of 20 μL. During the operation, ensure that there are no air bubbles and no contamination.

[0010] Furthermore, the NPM1-A primer mixture includes forward and reverse primers with a final primer concentration of 10 μM and an NPM1-A-Probe concentration of 10 μM. The ABL1 primer mixture includes forward and reverse primers with a final primer concentration of 10 μM and an ABL1-Probe concentration of 10 μM.

[0011] Furthermore, in step S6, the template nucleic acid and reaction mixture are loaded into the droplet generator, 70 μL of droplet generating oil is added to the oil hole at the bottom of the droplet generating card, and the prepared reaction mixture is added to the eight sample wells in the middle of the droplet generating card. After sealing with a rubber gasket, the assembled cartridge is inserted into the droplet generator, and the device is started to divide the reaction system into 20,000 uniform droplets using microfluidic technology.

[0012] Furthermore, in step S7, the generated oil droplets are carefully transferred to a 96-well PCR reaction plate, and the plate is sealed with an aluminum film using a PX1™ PCR Plate Sealer to ensure the reaction system is completely sealed. The sealed reaction plate is then placed in a thermal cycler and the amplification reaction is performed according to the following procedure: 95°C pre-denaturation for 10 min, 95°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 30 s, for a total of 40 cycles, followed by a final extension at 98°C for 10 min, and then placement at 4°C.

[0013] Furthermore, in step S9, the fluorescence signal data is statistically processed by analysis software. A one-dimensional or two-dimensional fluorescence scatter plot is automatically generated based on the intensity of the fluorescence signal in each microdroplet. The droplets are classified according to the fluorescence channel. Based on the blank control, negative control, and positive control, a threshold line is set at one-third of the distance between the signal intensities of negative and positive droplets in the one-dimensional fluorescence plot of NPM1 and ABL1. The results are exported in CSV format, and the NPM1 mutation expression level is calculated. When performing minimal residual disease analysis, the ABL1 copy number is ≥10,000. If the clinical sample cannot meet this standard due to differences in RNA expression at the maximum template amount, and the replicate results are consistent, it can still be judged as positive.

[0014] Compared with existing technologies, this method for dynamic monitoring of NPM1 mutations based on droplet digital PCR technology has the following advantages: I. This invention designs specific primers and fluorescent probes for type A mutations in exon 12 of the NPM1 gene, and uses ABL1 as an internal reference gene. Detection is performed using a droplet digital PCR platform. The amplification efficiency and specificity of the primers and probes are verified by real-time quantitative PCR. The combination of primer and probe concentrations is optimized using clinical samples, and cross-validation is performed using multiple samples to ensure the high sensitivity and specificity of this method for detecting NPM1 mutations. It can accurately detect trace amounts of NPM1 mutations in patients with acute myeloid leukemia, providing a reliable basis for efficacy evaluation, prognosis, and treatment decisions.

[0015] Second, this invention details the complete operational process from sample RNA extraction to data analysis and result interpretation, ensuring a high degree of standardization and repeatability throughout the entire detection process. Furthermore, regarding data analysis and result interpretation, the invention utilizes supporting analysis software to generate fluorescence scatter plots, appropriately sets threshold lines, and clarifies the calculation method for NPM1 mutation expression levels and the criteria for determining minimal residual disease analysis. This ensures the accuracy and reliability of the test results, helping clinicians to more accurately understand patients' conditions.

[0016] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0018] Figure 1 This is a flowchart of a method for dynamic monitoring of NPM1 mutations based on droplet digital PCR technology; Figure 2 Schematic diagram of primer and probe design for NPM1 and ABL1; Figure 3 A schematic diagram of a negative result from a dynamic monitoring method for NPM1 mutations based on droplet digital PCR technology. Figure 4 A schematic diagram of positive results from the NPM1 mutation dynamic monitoring method based on droplet digital PCR technology. Detailed Implementation

[0019] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0020] The purpose of this invention is to overcome the shortcomings of existing NPM1 mutation detection technologies, such as insufficient sensitivity, poor specificity, low repeatability, and dependence on standard curves. This invention provides a dynamic monitoring method for NPM1 mutations based on droplet digital PCR technology. This method uses RNA as a template to obtain cDNA via reverse transcription, designs specific primers and fluorescent probes targeting type A mutations in exon 12 of the NPM1 gene, and uses ABL1 as an internal reference gene. The reaction system is divided into numerous uniform droplets for independent amplification using a droplet digital PCR platform. This method exhibits high sensitivity, high specificity, and good repeatability, achieving absolute quantification without relying on a standard curve. It can accurately detect extremely low levels of NPM1 type A mutations in patients with acute myeloid leukemia (AML), simulating the pathological monitoring process of minimal residual disease in human AML, and improving the accuracy and reliability of dynamic monitoring of NPM1 mutations. To achieve the above objectives, this invention provides a method for dynamic monitoring of NPM1 mutations based on droplet digital PCR technology, such as... Figure 1As shown, it includes the following steps: Following the operating procedures in the instructions of the selected RNA extraction kit, extract nucleic acid RNA from the sample. After extraction, store the RNA at -80℃ for later use. This operation can maximize the preservation of RNA integrity, avoid RNA degradation affecting subsequent reverse transcription and amplification, and provide a high-quality template for subsequent experiments.

[0021] According to the instructions of the selected reverse transcription kit, the reaction system was prepared and the corresponding reaction program was set. The extracted RNA was reverse transcribed into cDNA. After the reverse transcription was completed, the cDNA was stored at -20℃ for later use. Reverse transcription converts RNA into more stable cDNA, which can improve the stability and accuracy of subsequent PCR reactions, and also facilitates long-term sample preservation and multiple tests.

[0022] Specific amplification primers and fluorescent probes were designed to target the type A mutation (c.860_863dupTCTG) in exon 12 of the NPM1 gene, such as... Figure 2 As shown, the forward primer is located in exon 10, the reverse primer spans the mutation insertion region, and the probe covers the junction region between exons 10 and 11. ABL1 is set as an internal reference gene, with its forward primer spanning exons 2 and 3, its reverse primer located in exon 3, and the probe positioned between the two primers. This design ensures that the primers and probe bind specifically to the target sequence only, effectively avoiding non-specific amplification and improving the specificity and sensitivity of the detection. The internal reference gene setting can correct for errors in sample processing and amplification, ensuring the reliability of the quantitative results.

[0023] After the design was completed, the amplification efficiency and specificity of NPM1 type A mutation and ABL1 primers and probes were first verified by real-time quantitative PCR (qPCR). Then, using clinically derived bone marrow and peripheral blood samples (positive and negative), the primer and probe concentration combinations were systematically optimized to evaluate their impact on amplification efficiency, background signal, and system stability, and to determine the optimal ddPCR reaction conditions. Further cross-validation was carried out using NPM1 RNA standards, HL60 cell lines, and clinical samples. Through validation and optimization, it was ensured that the primers and probes had good amplification performance, reduced background signal interference, and that the reaction system remained stable under different sample types and experimental conditions, thus ensuring the accuracy of subsequent detection results.

[0024] Mix the optimized concentration of specific primers and probes, ddPCR buffer, and nuclease-free water in the predetermined ratio to prepare a reaction mixture with a total volume of 20 μL. The components and amounts are as follows: Droplet PCR Supermix for Probe 10 μL, NPM1-A primer mixture (forward / reverse, final primer concentration 10 μM) 2 μL, NPM1-A-Probe (concentration 10 μM) 0.5 μL, ABL1 primer mixture (forward / reverse, final primer concentration 10 μM) 2 μL, ABL1-Probe (concentration 10 μM) 0.5 μL, template cDNA 25-250 ng, and nuclease-free water to a final volume of 20 μL. During the operation, ensure that there are no air bubbles and no contamination. Precise reaction system preparation can ensure that the proportion of each component is appropriate, which is conducive to the efficient PCR amplification reaction. Avoiding air bubbles and contamination can reduce experimental errors and false positive results.

[0025] The template nucleic acid and reaction mixture were loaded into the QX200 Droplet Generator. 70 μL of droplet generation oil for probes was added to the oil wells at the bottom of the droplet generation card. Then, the prepared reaction mixture was added to the eight sample wells in the middle of the droplet generation card. The card was then sealed with a rubber gasket. The assembled cartridge was inserted into the QX200 Droplet Generator, and the device was started to generate oil droplets. The reaction system was divided into approximately 20,000 uniform droplets using microfluidic technology. The generation of a large number of uniform droplets can randomly distribute nucleic acid molecules into different droplets, achieving single-molecule-level amplification and detection, significantly improving the detection sensitivity, and enabling the detection of low-abundance NPM1 mutations.

[0026] The generated oil droplets were carefully transferred to a 96-well PCR plate and sealed with an aluminum foil using a PX1™ PCR Plate Sealer to ensure the reaction system was completely sealed. The sealed plate was then placed in a thermal cycler and the amplification reaction was performed according to the following procedure: 95°C pre-denaturation for 10 min, 95°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 30 s, for a total of 40 cycles, followed by a final extension at 98°C for 10 min, and then incubation at 4°C. Strict sealing procedures prevent evaporation of the reaction solution and cross-contamination. A reasonable PCR amplification program ensures sufficient denaturation of the DNA template, effective annealing of the primers, and efficient extension of the DNA strand, ensuring the specificity and efficiency of the amplification reaction and providing sufficient amplification products for subsequent droplet detection.

[0027] After PCR amplification, the reaction plate is placed in the QX200 Droplet Reader for fluorescence signal reading. The QuantaSoft software platform is used for data acquisition and analysis. The sample well position information is entered in the software, and the fluorescence channels corresponding to NPM1 (FAM channel) and ABL1 (VIC channel) are selected. The signal reading program is started. The droplet reader can accurately identify the fluorescence signal of each droplet. Through different fluorescence channels, NPM1 mutation and internal reference gene ABL1 can be detected simultaneously, realizing the synchronous quantification of both and improving detection efficiency and accuracy.

[0028] The fluorescence signal data is statistically processed using the accompanying analysis software (QuantaSoft). The system automatically generates one-dimensional or two-dimensional fluorescence scatter plots based on the intensity of the fluorescence signal within each microdroplet, and classifies the droplets according to fluorescence channels (FAM corresponds to NPM1, VIC corresponds to ABL1). Based on blank control (NTC), negative control, and positive control, the one-dimensional fluorescence plots of NPM1 and ABL1 are analyzed. In the Plot, a threshold line is set at one-third of the distance between the signal intensities of negative and positive droplets. Results are exported in CSV format. The detection range for a single well is 30,000–100,000 copies. The final NPM1 mutation expression level is calculated as (NPM1 mutation copy number / ABL1 copy number) × 100%. For minimal residual disease (MRD) analysis, the ABL1 copy number must reach ≥10,000. If clinical samples cannot meet this standard due to differences in RNA expression at the maximum template amount (250 ng), and the replicate results are consistent, they can still be considered positive. This data analysis and interpretation method can accurately distinguish between positive and negative droplets. The precise quantification of NPM1 mutation is achieved through internal reference gene correction. Strict interpretation criteria ensure the reliability of the results and can sensitively detect minimal residual disease, providing an accurate basis for clinical diagnosis and treatment.

[0029] The following example illustrates the dynamic monitoring of NPM1 mutations based on droplet digital PCR technology: One bone marrow sample and one peripheral blood sample were selected from patients with acute myeloid leukemia (AML) (confirmed by previous testing to contain NPM1 A mutation). Another peripheral blood sample from a healthy person was used as a negative control. Total RNA was extracted from the samples using an RNA extraction kit (operated according to the instructions). The RNA concentration and purity were detected by ultraviolet spectrophotometer (A260 / A280 ratio controlled between 1.8 and 2.0). The samples were frozen and stored at -80℃ for later use.

[0030] Take 1 μg of extracted RNA and prepare a 20 μL reaction system using a reverse transcription kit: 4 μL of 5× reverse transcription buffer, 2 μL of 10 mM dNTP mixture, 1 μL of reverse transcriptase, 1 μL of random primers, 1 μg of RNA template, and make up the volume with nuclease-free water. The reaction program is as follows: incubate at 25℃ for 10 min, reverse transcribe at 42℃ for 60 min, and terminate the reaction by heating at 70℃ for 15 min. Store the obtained cDNA at -20℃.

[0031] All primers and probes are shown below: Primer sequences: Probe sequence: The total volume of the digital PCR (ddPCR) reaction system used in this invention is 20 μL, and the components and amounts are as follows: Droplet PCR Supermix for Probe: 10μL; NPM1-A primer mixture (forward / reverse, final primer concentration 10 μM): 2 μL; NPM1-A-Probe (concentration 10μM): 0.5μL; ABL1 primer mixture (forward / reverse, final primer concentration 10 μM): 2 μL; ABL1-Probe (concentration 10μM): 0.5μL; Template cDNA: 25-250 ng; Nuclease-free water (ddH2O): Add to a total reaction volume of 20 μL.

[0032] After the reaction mixture is prepared, it is gently mixed and centrifuged to proceed to the next step of oil droplet generation.

[0033] Droplet generation was performed using the Bio-Rad QX200 Droplet Generator. The procedure was as follows: Place a brand-new DG8 Cartridge into the Droplet Generator Holder and install the droplet generation card. Add 70 μL of droplet generation oil for probes to the oil wells at the bottom of the droplet generation card. Then, add the prepared reaction mixture to the eight sample wells in the middle of the droplet generation card and seal with a rubber gasket. Insert the assembled cartridge into the QX200 Droplet Generator, start the device to generate oil droplets, and then proceed to the PCR amplification step.

[0034] After the oil droplets are generated, the droplets are carefully transferred to a 96-well PCR reaction plate and heat-sealed with PX1™ PCR PlateSealer aluminum film to ensure the reaction system is completely sealed. The sealed PCR plate is then placed in a thermal cycler and PCR amplification is performed according to the following program: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 40 cycles, 98℃ final extension for 10 min, and incubation at 4℃.

[0035] After PCR amplification, the reaction plate is placed into the QX200 Droplet Reader for fluorescence signal reading. The QuantaSoft software platform is used for data acquisition and analysis. The sample well position information is entered in the software, the corresponding fluorescence channel is selected (FAM corresponds to NPM1, VIC corresponds to ABL1), and the signal reading program is started.

[0036] After signal acquisition is complete, click the "Analyze" button in the QuantaSoft software to start the data analysis program. The system will automatically generate a one-dimensional or two-dimensional fluorescence scatter plot based on the intensity of the fluorescence signal within each microdroplet, and classify the droplets according to the fluorescence channel (FAM corresponds to NPM1, VIC corresponds to ABL1), identifying mutant, wild-type, and negative droplets respectively. Based on the blank control (NTC), negative control, and positive control, the one-dimensional fluorescence plot (1D) of NPM1 and ABL1 is used. In AmplitudePlot, set a threshold line (one-third of the distance between the negative and positive droplet signal intensities) and export the results in CSV format. The detection range for a single well is 30,000–100,000 copies. The final NPM1 mutation expression level is calculated as (NPM1 mutation copy number / ABL1 copy number) × 100%. When performing minimal residual disease (MRD) analysis, the ABL1 copy number must reach ≥10,000. If clinical samples cannot meet this standard due to differences in RNA expression at the maximum template amount (250 ng), and the replicate results are consistent, they can still be judged as positive.

[0037] The following is an example of a typical two-dimensional scatter plot for negative and positive results in ddPCR detection: like Figure 3 As shown, no obvious blue (representing NPM1 mutation signal) or orange (representing simultaneous positivity of NPM1 and ABL1) oil droplet signals were observed in the scatter plot. Only background droplets (negative signal and ABL1 internal reference signal) were observed, which was determined to be negative. like Figure 4 As shown, the scatter plot shows both distinct blue oil droplets (representing NPM1 mutation signals) and orange oil droplets (representing simultaneous positivity for NPM1 and ABL1), indicating a positive result.

[0038] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for dynamic monitoring of NPM1 mutations based on droplet digital PCR technology, characterized in that, The method includes the following specific steps: S1. Extract RNA from the sample; S2. The extracted RNA is reverse transcribed into cDNA; S3. Design specific primers and fluorescent probes for type A mutation in exon 12 of the NPM1 gene, and design primers and fluorescent probes for the ABL1 gene. S4. Verify the designed primers and probes and confirm the performance of the reaction system; S5. Prepare the reaction mixture; S6. Generate oil droplets from the reaction mixture; S7. Perform PCR amplification on the system after oil droplets are generated; S8. Detect PCR amplification products using a droplet reader; S9. Analyze the test data and interpret the results.

2. The method for dynamic monitoring of NPM1 mutations based on droplet digital PCR technology according to claim 1, characterized in that, In step S3, highly specific amplification primers and fluorescent probes are designed for the type A mutation in exon 12 of the NPM1 gene. The forward primer is located in exon 10, the reverse primer spans the mutation insertion region, and the probe covers the connection region between exons 10 and 11.

3. The method for dynamic monitoring of NPM1 mutations based on droplet digital PCR technology according to claim 1, characterized in that, In step S3, ABL1 is set as an internal reference gene, with its forward primer spanning exons 2 and 3, its reverse primer located in exon 3, and the probe located between the two primers.

4. The method for dynamic monitoring of NPM1 mutations based on droplet digital PCR technology according to claim 1, characterized in that, In step S4, the amplification efficiency and specificity of NPM1 type A mutation and ABL1 primers and probes are verified by real-time quantitative PCR. Then, clinical bone marrow and peripheral blood samples, including positive and negative samples, are used to optimize the primer and probe concentration combination and evaluate its impact on amplification efficiency, background signal and system stability. The optimal ddPCR reaction conditions are determined. Furthermore, cross-validation is carried out using NPM1 RNA standard, HL60 cell line and clinical samples to confirm the performance of the system.

5. The method for dynamic monitoring of NPM1 mutations based on droplet digital PCR technology according to claim 1, characterized in that, In step S4, the optimized concentration of specific primers and probes, ddPCR buffer, and nuclease-free water are mixed in a predetermined ratio to prepare a reaction mixture with a total volume of 20 μL. The components and amounts are as follows: Droplet PCR Supermix for Probe 10 μL, NPM1-A primer mixture 2 μL, NPM1-A-Probe 0.5 μL, ABL1 primer mixture 2 μL, ABL1-Probe 0.5 μL, template cDNA 25-250 ng, and nuclease-free water to a final volume of 20 μL. During the operation, ensure that there are no air bubbles and no contamination.

6. The method for dynamic monitoring of NPM1 mutations based on droplet digital PCR technology according to claim 5, characterized in that, The NPM1-A primer mixture includes forward and reverse primers with a final primer concentration of 10 μM and an NPM1-A-Probe concentration of 10 μM. The ABL1 primer mixture includes forward and reverse primers with a final primer concentration of 10 μM and an ABL1-Probe concentration of 10 μM.

7. The method for dynamic monitoring of NPM1 mutations based on droplet digital PCR technology according to claim 1, characterized in that, In step S6, the template nucleic acid and reaction mixture are loaded into the droplet generator, 70 μL of droplet generating oil is added to the oil hole at the bottom of the droplet generating card, and the prepared reaction mixture is added to the 8 sample wells in the middle of the droplet generating card. After sealing with a rubber gasket, the assembled cartridge is inserted into the droplet generator, and the device is started to divide the reaction system into 20,000 uniform droplets through microfluidic technology.

8. The method for dynamic monitoring of NPM1 mutations based on droplet digital PCR technology according to claim 1, characterized in that, In step S7, the generated oil droplets are carefully transferred to a 96-well PCR reaction plate, and the plate is sealed with aluminum film using a PX1™ PCR PlateSealer to ensure the reaction system is completely sealed. The sealed reaction plate is then placed in a thermal cycler and the amplification reaction is performed according to the following procedure: 95°C pre-denaturation for 10 min, 95°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 30 s, for a total of 40 cycles, 98°C final extension for 10 min, and 4°C incubation.

9. The method for dynamic monitoring of NPM1 mutations based on droplet digital PCR technology according to claim 1, characterized in that, In step S9, the fluorescence signal data is statistically processed by analysis software. A one-dimensional or two-dimensional fluorescence scatter plot is automatically generated based on the intensity of the fluorescence signal in each microdroplet. The droplets are classified according to the fluorescence channel. Based on the blank control, negative control, and positive control, a threshold line is set at one-third of the distance between the signal intensities of negative and positive droplets in the one-dimensional fluorescence plot of NPM1 and ABL1. The results are exported in CSV format. The mutation expression level of NPM1 is calculated. When performing minimal residual disease analysis, the ABL1 copy number is ≥10,000. If the clinical sample cannot meet this standard due to differences in RNA expression at the maximum template amount, and the replicate results are consistent, it can still be judged as positive.

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