Primer probe group for detecting mutation of bZIP region of CEBPA gene, kit and drop-offset dPCR method

Abstract

The invention relates to the technical field of biology, in particular to a primer probe set for detecting mutation of a bZIP region of a CEBPA gene, a kit and a drop-offdPCR method. The primer and the probe are designed according to the DNA sequence of the CEBPA gene, and cover the area where common in-frame mutation sites in the bZIP area of the gene are located. When mutation exists in the bZIP region of the CEBPA gene, the wild-type probe located at the mutation site cannot be tightly combined with the template, and the other three wild-type probes located outside the mutation site in the same group can still be combined with the template, so that mutation of multiple sites can be detected through one experiment. The kit disclosed by the invention can be used for detecting in-frame insertion / deletion mutations including K304Q305insL, Q305dup, K313V314insE and the like in the bZIP region of the CEBPA gene, has relatively high sensitivity, and can be used for MRD (Magnetic Resonance Diffraction) monitoring.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a primer probe set, a kit and a drop-off ddPCR method for detecting mutations in the bZIP region of a CEBPA gene. Background Art

[0002] The transcription factor CCAAT / enhancer binding protein α (CEBPA) gene is located on human chromosome 19q13.1 and mainly consists of two parts: the N-terminal transcription activation domain (TAD) and the C-terminal basic region leucine zipper domain (bZIP).

[0003] CEBPA mutations are among the most common genetic mutations in acute myeloid leukemia (AML). The detection rate of CEBPA mutations in primary AML patients is 4%-20%, with the vast majority presenting as biallelic mutations. Classic CEBPA biallelic mutations include a frameshift mutation at the N-terminus and an in-frame mutation at the C-terminus. Based on advances in sequencing technology and the conduct of large-sample clinical studies, the ELN (2022) guidelines and the fifth edition of the WHO guidelines include in-frame mutations in the CEBPA bZIP region as independent factors influencing a favorable prognosis in AML patients. Despite this, retrospective studies have shown that the cumulative relapse rate in patients with this mutation still exceeds 40%. Therefore, minimal residual disease (MRD) monitoring has important clinical value in the prognostic assessment of patients with CEBPA mutations.

[0004] Currently, clinical methods for monitoring MRD in gene mutations primarily include Sanger sequencing, next-generation sequencing (NGS), and real-time quantitative PCR. However, mutations in the CEBPA bZIP domain are highly heterogeneous, lack clear mutation hotspots, and have a wide distribution of mutation sites. Sanger sequencing technology is unsuitable for MRD monitoring due to its low sensitivity (≥20%). While NGS can achieve comprehensive detection, its high cost and long detection cycle limit its application in dynamic MRD monitoring. Real-time quantitative PCR is also unsuitable for MRD monitoring due to its inability to achieve absolute quantification. While digital PCR (ddPCR) offers the advantage of high sensitivity, its limited ability to detect a single mutation type and its applicability to only known mutations makes it difficult to address the complexities of CEBPA gene mutations. Traditional ddPCR methods, while highly sensitive, detect a relatively limited number of mutations and are limited to known mutations, making them unsuitable for detecting CEBPA mutations. Furthermore, the high GC content (>70%) of the CEBPA bZIP region results in low amplification efficiency using traditional PCR, further complicating detection difficulties. Therefore, the detection of mutations in the CEBPA bZIP region of patients needs to be further optimized. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention provides a primer-probe set, a kit, and a drop-off ddPCR method for detecting mutations in the bZIP region of the CEBPA gene. The primer set and probe provided by the present invention are designed based on the DNA sequence of the CEBPA gene, offering high specificity. Only one pair of universal primers and two sets of probes are required to cover the bZIP region and detect common in-frame insertion / deletion mutations located in the bZIP region. The drop-off ddPCR method for detecting CEBPA gene mutations provided by the present invention has a minimum detection limit of 0.101%, demonstrating high sensitivity.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a primer probe set for detecting mutations in the bZIP region of the CEBPA gene, wherein the primer probe set comprises a specific primer pair and a probe set for detecting mutation sites in the bZIP region of the CEBPA gene;

[0008] The nucleotide sequences of the specific primer pairs are shown in SEQ ID NO. 1-2;

[0009] The nucleotide sequences of the probe group are shown in SEQ ID NOs. 3 to 6 or SEQ ID NOs. 7 to 10.

[0010] Preferably, the 5' end of the probe in the probe group is connected to a fluorescent group, and the 3' end is connected to a quenching group.

[0011] Preferably, the fluorescent group is one or more of FAM, VIC, HEX, TRT, CY3, CY5, ROX and JOE, and the quencher group is one or more of TAMRA, DABCYL, MGB, BHQ-1, BHQ-2 and BHQ-3.

[0012] Preferably, the mutations in the bZIP region of the CEBPA gene are in-frame insertion / deletion mutations including K304_Q305insL, Q305dup, K313_V314insE, E316_L317insR, L317dup, E309dup, E309_T310insK, Q312dup, K313dup, and K313del.

[0013] The present invention also provides a kit for detecting mutations in the bZIP region of the CEBPA gene for non-diagnostic purposes, comprising the above primer probe set.

[0014] Preferably, the kit further comprises a CEBPA mutant plasmid as a positive control and a wild-type plasmid as a negative control.

[0015] The present invention also provides a drop-off ddPCR method for quantitatively detecting mutations in the bZIP region of the CEBPA gene for non-diagnostic purposes, comprising the following steps:

[0016] (1) Extracting DNA from the sample to be tested;

[0017] (2) mixing the DNA of the sample to be tested with the components in the kit to obtain a ddPCR reaction mixture;

[0018] (3) After mixing the ddPCR reaction mixture with the oil phase, the resulting mixture is prepared into microreaction droplets using a microdroplet preparation instrument;

[0019] (4) After the micro-reaction droplets are introduced into the chip, a PCR amplification reaction is performed, and signals of the micro-droplets in the chip after the PCR amplification reaction are collected to calculate the allele mutation frequency of the CEBPA gene in the sample to be tested.

[0020] Preferably, the sample to be tested in step (1) is the patient's peripheral blood, bone marrow tissue or tumor tissue.

[0021] Preferably, the ddPCR reaction mixture in step (2) comprises the following components:

[0022] 10-20 μl of 2×dPCR reaction buffer, 0.4-0.6 μl of SNUPP DNA polymerase, 22-26 pmol each of the specific primers shown in SEQ ID NOs. 1-2, 2-4 pmol each of the probes shown in SEQ ID NOs. 3-6 or SEQ ID NOs. 7-10, 40-500 ng of the test sample DNA, and nuclease-free water to make up to 30 μl.

[0023] Preferably, the PCR amplification reaction procedure in step (4) is: 50°C for 5 min, 95°C for 5 min, 96°C for 15 s, 60-65°C for 30 s, 40-50 cycles, and finally maintained at 25°C.

[0024] Preferably, a set of probes containing FAM labeling, VIC labeling, ROX labeling, and CY5 labeling are used in the same PCR reaction system. According to the results of automatic analysis by the droplet analyzer, the copy number concentrations of the FAM, VIC, ROX, and CY5 channels in the sample are read to obtain the mutation load level of the CEBPA gene in the sample.

[0025] Preferably, the CEBPA gene allele frequency (VAF) is calculated as:

[0026]

[0027] Preferably, the limit of blank (LOB) and the lowest limit of detection (LOD) of each mutation type of the CEBPA gene are shown in Table 1 below:

[0028] Table 1 Blank limits and minimum detection limits for each mutation type of CEBPA gene

[0029]

[0030]

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) The present invention provides a detection kit for detecting CEBPA gene mutations based on drop-off ddPCR, wherein the primers and probes are designed according to the DNA sequence of the CEBPA gene and have strong specificity.

[0033] (2) The present invention provides a method for detecting CEBPA gene mutations based on drop-off ddPCR. Only one pair of universal primers and two sets of probes are needed for the CEBPA gene to cover the bZIP region and detect common in-frame insertion / deletion mutations located in the bZIP region.

[0034] (3) The detection method for detecting CEBPA gene mutation based on drop-off ddPCR provided by the present invention has a minimum detection limit of 0.101% and high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0036] Figure 1 This is a graph showing the drop-off ddPCR test results for a bone marrow sample from a patient with a negative CEBPA gene mutation in Example 1; the X-axis represents the fluorescence signal value of FAM, the Y-axis represents the fluorescence signal value of VIC, the upper right quadrant represents wild-type droplets, and the lower left quadrant represents double-negative droplets;

[0037] Figure 2 This is a graph showing the drop-off ddPCR test results for a bone marrow sample from a patient with a negative CEBPA gene mutation in Example 1; the X-axis represents the fluorescence signal value of ROX, the Y-axis represents the fluorescence signal value of CY5, the upper right quadrant represents wild-type droplets, and the lower left quadrant represents double-negative droplets;

[0038] Figure 3 This figure shows the drop-off ddPCR test results for a bone marrow sample from a patient with the CEBPA gene Q312dup mutation in Example 2. The X-axis represents the fluorescence signal value of FAM, the Y-axis represents the fluorescence signal value of VIC, the upper right quadrant represents wild-type droplets, and the lower left quadrant represents double-negative droplets.

[0039] Figure 4 This figure shows the drop-off ddPCR test results for a bone marrow sample from a patient with the CEBPA gene Q312dup mutation in Example 2. The X-axis represents the fluorescence signal value of ROX, the Y-axis represents the fluorescence signal value of CY5, the upper left quadrant represents CEBPA Q312dup mutant droplets, the upper right quadrant represents wild-type droplets, and the lower left quadrant represents double-negative droplets.

[0040] Figure 5 Figure 3 shows the drop-off ddPCR test results for a bone marrow sample from a patient with the CEBPA gene K313dup mutation. The X-axis represents the fluorescence signal value of FAM, the Y-axis represents the fluorescence signal value of VIC, the upper right quadrant represents wild-type droplets, and the lower left quadrant represents double-negative droplets.

[0041] Figure 6This figure shows the drop-off ddPCR test results for bone marrow samples from patients with the CEBPA gene K313dup mutation in Example 3; the X-axis represents the fluorescence signal value of ROX, the Y-axis represents the fluorescence signal value of CY5, the upper left quadrant represents CEBPAK313dup mutant droplets, the upper right quadrant represents wild-type droplets, and the lower left quadrant represents double-negative droplets;

[0042] Figure 7 This is a graph showing the drop-off ddPCR test results for a bone marrow sample from a patient with the CEBPA gene K304_Q305insL mutation in Example 4; the X-axis represents the fluorescence signal value of FAM, the Y-axis represents the fluorescence signal value of VIC, the upper right quadrant represents wild-type droplets, and the lower left quadrant represents double-negative droplets;

[0043] Figure 8 This is a graph showing the drop-off ddPCR test results for a bone marrow sample from a patient with the CEBPA gene K304_Q305insL mutation in Example 4; the X-axis represents the fluorescence signal value of ROX, the Y-axis represents the fluorescence signal value of CY5, the upper left quadrant represents CEBPAK304_Q505insL mutant droplets, the upper right quadrant represents wild-type droplets, and the lower left quadrant represents double-negative droplets;

[0044] Figure 9 This figure shows the drop-off ddPCR test results for a bone marrow sample from a patient with the CEBPA gene L317dup mutation in Example 5. The X-axis represents the fluorescence signal value of FAM, the Y-axis represents the fluorescence signal value of VIC, the upper right quadrant represents wild-type droplets, and the lower left quadrant represents double-negative droplets.

[0045] Figure 10 This figure shows the drop-off ddPCR test results for a bone marrow sample from a patient with the CEBPA gene L317dup mutation in Example 5; the X-axis represents the fluorescence signal value of ROX, the Y-axis represents the fluorescence signal value of CY5, the lower right quadrant represents CEBPAL317dup mutant droplets, the upper right quadrant represents wild-type droplets, and the lower left quadrant represents double-negative droplets;

[0046] Figure 11 This figure shows the drop-off ddPCR test results for a bone marrow sample from a patient with the CEBPA gene Q305dup mutation in Example 6; the X-axis represents the fluorescence signal value of FAM, the Y-axis represents the fluorescence signal value of VIC, the upper right quadrant represents wild-type droplets, and the lower left quadrant represents double-negative droplets;

[0047] Figure 12This figure shows the drop-off ddPCR test results for a bone marrow sample from a patient with the CEBPA gene Q305dup mutation in Example 6; the X-axis represents the fluorescence signal value of ROX, the Y-axis represents the fluorescence signal value of CY5, the upper left quadrant represents CEBPA Q305dup mutant droplets, the upper right quadrant represents wild-type droplets, and the lower left quadrant represents double-negative droplets;

[0048] Figure 13 This figure shows the drop-off ddPCR test results for a bone marrow sample from a patient with the CEBPA gene E309dup mutation in Example 7. The X-axis represents the fluorescence signal value of FAM, the Y-axis represents the fluorescence signal value of VIC, the upper right quadrant represents wild-type droplets, and the lower left quadrant represents double-negative droplets.

[0049] Figure 14 This figure shows the drop-off ddPCR test results for a bone marrow sample from a patient with the CEBPA gene E309dup mutation in Example 7; the X-axis represents the fluorescence signal value of ROX, the Y-axis represents the fluorescence signal value of CY5, the upper left quadrant represents CEBPAE309dup mutant droplets, the upper right quadrant represents wild-type droplets, and the lower left quadrant represents double-negative droplets;

[0050] Figure 15 This is a graph showing the drop-off ddPCR test results for a bone marrow sample from a patient with the CEBPA gene E309_T310insK mutation in Example 8; the X-axis represents the fluorescence signal value of FAM, the Y-axis represents the fluorescence signal value of VIC, the upper right quadrant represents wild-type droplets, and the lower left quadrant represents double-negative droplets;

[0051] Figure 16 This is a graph showing the drop-off ddPCR test results for a bone marrow sample from a patient with the CEBPA gene E309_T310insK mutation in Example 8; the X-axis represents the fluorescence signal value of ROX, the Y-axis represents the fluorescence signal value of CY5, the upper left quadrant represents CEBPAE309dup_T310insK mutant droplets, the upper right quadrant represents wild-type droplets, and the lower left quadrant represents double-negative droplets;

[0052] Figure 17 This figure shows the drop-off ddPCR test results for a bone marrow sample from a patient with the CEBPA gene K313del mutation in Example 9; the X-axis represents the fluorescence signal value of FAM, the Y-axis represents the fluorescence signal value of VIC, the upper right quadrant represents wild-type droplets, and the lower left quadrant represents double-negative droplets;

[0053] Figure 18This is a graph showing the drop-off ddPCR test results for a bone marrow sample from a patient with the CEBPA gene K313del mutation in Example 9; the X-axis represents the fluorescence signal value of ROX, the Y-axis represents the fluorescence signal value of CY5, the upper left quadrant represents CEBPA K313del mutant droplets, the upper right quadrant represents wild-type droplets, and the lower left quadrant represents double-negative droplets;

[0054] Figure 19 This is a graph showing the drop-off ddPCR test results for a bone marrow sample from a patient with the CEBPA gene K313_V314insE mutation in Example 10; the X-axis represents the fluorescence signal value of FAM, the Y-axis represents the fluorescence signal value of VIC, the upper right quadrant represents wild-type droplets, and the lower left quadrant represents double-negative droplets;

[0055] Figure 20 This is a graph showing the drop-off ddPCR test results for a bone marrow sample from a patient with the CEBPA gene K313_V314insE mutation in Example 10; the X-axis represents the fluorescence signal value of ROX, the Y-axis represents the fluorescence signal value of CY5, the lower right quadrant represents CEBPAK313_V314insE mutant droplets, the upper right quadrant represents wild-type droplets, and the lower left quadrant represents double-negative droplets;

[0056] Figure 21 This is a graph showing the drop-off ddPCR test results for a bone marrow sample from a patient with the CEBPA gene E316_L317insR mutation in Example 11; the X-axis represents the fluorescence signal value of FAM, the Y-axis represents the fluorescence signal value of VIC, the upper right quadrant represents wild-type droplets, and the lower left quadrant represents double-negative droplets;

[0057] Figure 22 This is a graph showing the drop-off ddPCR test results for a bone marrow sample from a patient with the CEBPA gene E316_L317insR mutation in Example 11; the X-axis represents the fluorescence signal value of ROX, the Y-axis represents the fluorescence signal value of CY5, the lower right quadrant represents CEBPAE316_L317insR mutant droplets, the upper right quadrant represents wild-type droplets, and the lower left quadrant represents double-negative droplets;

[0058] Figure 23 This is a linear graph of the drop-off ddPCR test results of CEBPA gene K304_Q305insL mutant samples at different concentrations;

[0059] Figure 24 This is a linear graph of the drop-off ddPCR test results of CEBPA gene Q305dup mutant samples with different concentrations;

[0060] Figure 25This is a linear graph of the drop-off ddPCR test results of samples with different concentrations of the E309dup mutant of the CEBPA gene;

[0061] Figure 26 This is a linear graph of the drop-off ddPCR test results of samples with different concentrations of the E309_T310insK mutant of the CEBPA gene;

[0062] Figure 27 This is a linear graph of the drop-off ddPCR test results of CEBPA gene Q312dup mutant samples with different concentrations;

[0063] Figure 28 This is a linear graph of the drop-off ddPCR test results of CEBPA gene K313dup mutant samples with different concentrations.

[0064] Figure 29 This is a linear graph of the drop-off ddPCR test results of CEBPA gene K313del mutant samples with different concentrations;

[0065] Figure 30 This is a linear graph of the drop-off ddPCR test results of CEBPA gene K313_V314insE mutant samples at different concentrations;

[0066] Figure 31 This is a linear graph of the drop-off ddPCR test results of samples with different concentrations of the E316_L317insR mutant of the CEBPA gene;

[0067] Figure 32 This is a linear graph of the drop-off ddPCR test results of CEBPA gene L317dup mutant samples with different concentrations. DETAILED DESCRIPTION

[0068] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0069] The experimental methods used in the following examples, unless otherwise specified, are conventional methods. Materials and reagents used in the following examples are commercially available unless otherwise specified. Because VIC and HEX have similar fluorescence wavelengths and can share a common fluorescence channel, the fluorescent group attached to the 5' end of the probes described in the following examples of the present invention is VIC, but the fluorescence is collected using the HEX channel.

[0070] The primer sets and probes provided by the present invention are designed based on the DNA sequence of the CEBPA gene. Two groups of four wild-type probes in each group are designed for the bZIP domain of the CEBPA gene (the probes shown in SEQ ID NOs. 3 to 6 are set as the first group of probes, and the probes shown in SEQ ID NOs. 7 to 10 are set as the second group of probes), thereby achieving the purpose of covering the CEBPA region from amino acids 278 to 325. When an insertion or deletion mutation occurs at a site in this region, the wild-type probe located at the mutation site cannot bind tightly to the template, while the wild-type probe outside the mutation site can still bind tightly to the template. Changes in the gene copy number concentration of the fluorescent channel corresponding to different probes can be detected by drop-off ddPCR.

[0071] The primer sequences shown in SEQ ID NO.1~2 are as follows:

[0072] CEBPAbZIP region forward primer: 5'-GGGCAAGGCCAAGAAGT-3'

[0073] CEBPAbZIP region reverse primer: 5′-GCGGCTCAGCTGTTCCAC-3′.

[0074] The probe sequences described in SEQ ID NO.3 to 10 are as follows:

[0075] CEBPAbZIP region probe group 1:

[0076] Probe 1: 5′-FAM-ACTCGTTGCTGTTCTTGTCCACC-BHQ1-3′

[0077] Probe 2: 5′-VIC-CACCGCGATGTTGTTGCGCTC-BHQ1-3′

[0078] Probe 3: 5′-ROX-CAAGGCCAAGCAGCGCAACGT-BHQ1-3′

[0079] Probe 4: 5'-CY5-CAGAAGGTGCTGGAGCTGACCA-BHQ2-3'

[0080] CEBPAbZIP region probe group 2:

[0081] Probe 1: 5′-FAM-AACGAGTACCGGGTGCGGCG-BHQ1-3′

[0082] Probe 2: 5′-VIC-TGCGCAAGAGCCGCGACAAG-BHQ1-3′

[0083] Probe 3: 5′-ROX-ACGTGGAGACGCAGCAGAAGGT-BHQ1-3′

[0084] Probe 4: 5′-CY5-AGTGACAATGACCGCCTGCGC-BHQ2-3′.

[0085] Example 1

[0086] Taking the bone marrow samples of patients with negative CEBPA gene mutation as an example, the detection method is explained in detail.

[0087] 1. Isolation of bone marrow mononuclear cells and DNA extraction

[0088] (1) Add 5 ml of red blood cell lysis buffer to the bone marrow sample and mix well, then let it stand for 1 min;

[0089] (2) Centrifuge at 12000 rpm for 20 seconds, discard the supernatant, and retain the precipitate;

[0090] (3) Add 900 μl of cell lysis buffer and mix thoroughly by pipetting;

[0091] (4) Add 4.5 μL of RNase, mix thoroughly by inversion, incubate at 37°C for 15 minutes, and then incubate on ice for 1 minute;

[0092] (5) Add 300 μL of protein precipitation solution and vortex to mix;

[0093] (6) Centrifugation at 13000 rpm for 1 minute;

[0094] (7) Take a new EP tube, draw 600 μL of isopropanol, add 600 μL of the supernatant after the above centrifugation to the isopropanol, and mix thoroughly by inversion;

[0095] (8) Centrifuge again at 13000 rpm for 1 min;

[0096] (9) Aspirate the supernatant, add 600 μl of 70% ethanol solution, and mix by inversion;

[0097] (10) Centrifuge again at 13000 rpm for 1 min;

[0098] (11) Discard the supernatant, dry the precipitate, add an appropriate amount of DNA dissolution solution, and incubate in a 65°C metal bath for 5 min;

[0099] (12) The absorbance (A) value of the DNA sample was measured by UV spectrophotometer, and the concentration was adjusted to 40 ng / μl and stored at 4°C.

[0100] 2. Using the above DNA template, prepare the ddPCR reaction mixture. The preparation ratio is as shown in Table 2:

[0101] Table 2 Preparation of ddPCR reaction mixture

[0102]

[0103]

[0104] The above reaction solutions were mixed evenly, vortexed for 30 seconds, and centrifuged briefly to collect the reaction solution at the bottom of the tube and placed on ice for later use.

[0105] 3. Prepare the oil phase mixture according to the proportions shown in Table 3 below:

[0106] Table 3 Preparation of oil phase mixture

[0107] Oil phase A 2ml Oil phase B 1ml

[0108] Prepare the reaction oil phase according to the table above (Oil phases A and B are both oil-based reagents for ddPCR instruments produced by Shanghai Little Turtle Technology Co., Ltd.) Use the mixed oil phase mixture within 30 minutes.

[0109] 4. Microdroplet Preparation

[0110] (1) Open the front panel door of the instrument, connect the BioDigital Loader Z200 sample processing system power, start the panel, place the digital PCR chip in the Loader Z200 chip compartment, add the above reaction system and oil phase mixture, close the door and complete the sample loading program settings according to the prompts on the instrument computer operation panel.

[0111] (2) After the injection is completed, remove the chip containing the droplets.

[0112] 5. PCR reaction

[0113] Place the chip in the chip slot of the Cycler Z200 PCR amplification instrument and perform the following reaction (see Table 4):

[0114] Table 4 PCR reaction program

[0115] 1 50℃ 5min 2 95℃ 5min 3 96℃ 15s 4 60-65℃ 30s 5 Goto3 For45times 6 25℃ Hold

[0116] 6. Chip reading and data analysis

[0117] After the PCR amplification reaction is completed, the chip is moved to the ImagerZ200 biochip reader and data analysis is performed based on the chip reading results. Figure 1-2Experimental results and related data. The results show that the concentrations of FAM, VIC, ROX, and CY5 in the sample DNA were 24,319.2 copies / μl, 24,319.2 copies / μl, and 24,319.2 copies / μl, respectively. The calculated allele frequency of the CEBPAbZIP region in the sample genome was 0%.

[0118] Example 2

[0119] Taking the bone marrow sample of a patient with CEBPA gene Q312dup mutation as an example, the specific detection process is the same as that in Example 1. After data analysis, the following can be obtained: Figure 3-4 Experimental results and related data. The sample DNA contained 10,786.1 copies / μl of FAM, 10,786.1 copies / μl of VIC, 9,419.8 copies / μl of ROX, and 10,786.1 copies / μl of CY5. The Q312dup mutation site was located in the second set of ROX fluorescent-labeled probes, probe 3. The calculated template concentration of the CEBPA gene Q312dup mutation was 1,366.3 copies / μl, and the CEBPA gene Q312dup allele mutation frequency was 12.667%.

[0120] Example 3

[0121] Taking the bone marrow sample of a patient with CEBPA gene K313dup mutation as an example, the specific detection process is the same as that in Example 1. After data analysis, the following can be obtained: Figure 5-6 Experimental results and related data. The sample DNA contained 8967.5 copies / μl of FAM, 8966.2 copies / μl of VIC, 4632.9 copies / μl of ROX, and 8967.5 copies / μl of CY5. The QK313dup mutation site was located in the second set of ROX fluorescent-labeled probes, probe 3. The calculated template concentration for the CEBPA gene K313dup mutation was 4334.17 copies / μl, and the CEBPA gene K313dup allele mutation frequency was 48.334%.

[0122] Example 4

[0123] Taking the bone marrow sample of a patient with CEBPA gene K304_Q305insL mutation as an example, the specific detection process is the same as that in Example 1. After data analysis, the following can be obtained: Figure 7-8Experimental results and related data. In the sample DNA, the FAM concentration was 13464.9 copies / μl, the VIC concentration was 13460.8 copies / μl, the ROX concentration was 13430.8 copies / μl, and the CY5 concentration was 13464.9 copies / μl. The K304_Q305insL mutation site was located in the second set of ROX fluorescent-labeled probes, probe 3. The calculated template concentration of the K304_Q305insL mutation in the CEBPA gene was 32.73 copies / μl, and the mutation frequency of the K304_Q305insL allele in the CEBPA gene was 24.310%.

[0124] Example 5

[0125] Taking the bone marrow sample of a patient with CEBPA gene L317dup mutation as an example, the specific detection process is the same as that in Example 1. After data analysis, the following can be obtained: Figure 9-10 Experimental results and related data. The sample DNA contained 12145.8 copies / μl of FAM, 12142.3 copies / μl of VIC, 12142.3 copies / μl of ROX, and 5851.6 copies / μl of CY5. The L317dup mutation site was located in probe 4 of the first set of CY5 fluorescent labels. The calculated template concentration for the CEBPA gene L317dup mutation was 6291.87 copies / μl, and the CEBPA gene L317dup allele mutation frequency was 51.813%.

[0126] Example 6

[0127] Taking the mixed sample of CEBPA gene Q305dup mutation and pure wild plasmid as an example, the specific detection process is the same as that in Example 1. After data analysis, the following can be obtained: Figure 11-12 Experimental results and related data. The sample DNA contained 9750.2 copies / μl of FAM, 9751.5 copies / μl of VIC, 5114.8 copies / μl of ROX, and 9751.5 copies / μl of CY5. The Q305dup mutation site was located in the first set of ROX fluorescent-labeled probes, probe 3. The calculated template concentration for the CEBPA gene Q305dup mutation was 4636.27 copies / μl, and the CEBPA gene Q305dup allele mutation frequency was 47.546%.

[0128] Example 7

[0129] Taking the mixed sample of CEBPA gene E309dup mutation and pure wild plasmid as an example, the specific detection process is the same as that in Example 1. After data analysis, the following can be obtained: Figure 13-14 Experimental results and related data. The sample DNA contained 9200.5 copies / μl of FAM, 9200.5 copies / μl of VIC, 5114.8 copies / μl of ROX, and 920.5 copies / μl of CY5. The E309dup mutation site was located in the second set of ROX fluorescent-labeled probes, probe 3. The calculated template concentration for the E309dup mutation in the CEBPA gene was 4492.90 copies / μl, and the mutation frequency of the E309dup allele in the CEBPA gene was 48.833%.

[0130] Example 8

[0131] Taking the mixed sample of CEBPA gene E309_T310insK mutation and pure wild plasmid as an example, the specific detection process is the same as that in Example 1. After data analysis, the following can be obtained: Figure 15-16 Experimental results and related data. The sample DNA contained 9068.9 copies / μl of FAM, 9068.9 copies / μl of VIC, 9056.3 copies / μl of ROX, and 9068.9 copies / μl of CY5. The E309_T310insK mutation site was located in the second set of ROX fluorescent-labeled probes, probe 3. The calculated template concentration for the E309_T310insK mutation in the CEBPA gene was 12.6 copies / μl, and the mutation frequency of the E309dup allele in the CEBPA gene was 0.139%.

[0132] Example 9

[0133] Taking the mixed sample of CEBPA gene K313del mutation and pure wild plasmid as an example, the specific detection process is the same as that in Example 1. After data analysis, the following can be obtained: Figure 17-18 Experimental results and related data. The sample DNA contained 9846.2 copies / μl of FAM, 9846.2 copies / μl of VIC, 7431.6 copies / μl of ROX, and 9846.2 copies / μl of CY5. The K313del mutation site was located in the second set of ROX fluorescent-labeled probes, probe 3. The calculated template concentration for the CEBPA gene K313del mutation was 2414.6 copies / μl, and the CEBPA gene K313del allele mutation frequency was 24.523%.

[0134] Example 10

[0135] Taking the mixed sample of CEBPA gene K313_V314insE mutation and pure wild plasmid as an example, the specific detection process is the same as that in Example 1. After data analysis, the following can be obtained: Figure 19-20 Experimental results and related data. The sample DNA contained 10,738.4 copies / μl of FAM, 10,748.9 copies / μl of VIC, 10,747.4 copies / μl of ROX, and 9,540.3 copies / μl of CY5. The K313_V314insE mutation site was located in probe 4 of the first set of CY5 fluorescent labels. The calculated template concentration for the K313_V314insE mutation in the CEBPA gene was 1,204.6 copies / μl, and the mutation frequency of the K313_V314insE allele in the CEBPA gene was 11.211%.

[0136] Example 11

[0137] Taking the mixed sample of CEBPA gene E316_L317insR mutation and pure wild plasmid as an example, the specific detection process is the same as that in Example 1. After data analysis, the following can be obtained: Figure 21-22 Experimental results and related data. The sample DNA contained 11231.9 copies / μl of FAM, 11239.9 copies / μl of VIC, 11243.1 copies / μl of ROX, and 11147.3 copies / μl of CY5. The E316_L317insR mutation site was located in the first set of ROX fluorescent-labeled probes, probe 4. The calculated template concentration for the E316_L317insR mutation in the CEBPA gene was 91.0 copies / μl, and the mutation frequency of the E316_L317insR allele in the CEBPA gene was 0.810%.

[0138] Example 12

[0139] ddPCR sensitivity experiments were performed using a pure wild-type plasmid of the CEBPA gene and CEBPA mutation-negative samples from different genomic sources. According to the method of Example 1, the CEBPA mutation-negative samples from different genomic sources were tested 20 times, and the limit of blank (LOB) was calculated as shown in Table 5:

[0140] Table 5 Blank limits for each mutation type of CEBPA gene

[0141] Mutation type SD LOB K304_Q305insL 0.028％ 0.089％ Q305dup 0.070％ 0.165％ E309dup 0.019％ 0.046％ E309_T310insK 0.019％ 0.046％ Q312dup 0.019％ 0.046％ K313dup 0.019％ 0.046％ K313del 0.019％ 0.046％ K313_V314insE 0.080％ 0.168％ E316_L317insR 0.092％ 0.185％ L317dup 0.092％ 0.185％

[0142] The pure mutant plasmids of each CEBPA mutant were spiked into the pure wild-type CEBPA plasmid at low concentrations. These samples were tested 20 times each, and the lower limit of detection (LOD) was calculated as shown in Table 6:

[0143] Table 6 Minimum detection limits for each mutation type of CEBPA gene

[0144] Mutation type SDs LOD K304_Q305insL 0.204％ 0.204％ Q305dup 0.375％ 0.375％ E309dup 0.101％ 0.101％ E309_T310insK 0.106％ 0.106％ Q312dup 0.101％ 0.101％ K313dup 0.146％ 0.146％ K313del 0.153％ 0.153％ K313_V314insE 0.400％ 0.400％ E316_L317insR 0.341％ 0.341％ L317dup 0.361％ 0.361％

[0145] According to the method of Example 1, pure mutant plasmids and wild-type plasmids of various CEBPA mutants were quantified, diluted, and mixed in different proportions to form samples with corresponding theoretical mutation concentrations. Each mutation concentration sample was tested three times, and the following data were obtained (see Tables 7 to 16):

[0146] Table 7 Drop-off ddPCR detection results of CEBPA gene K304_Q305insL mutant samples at different concentrations

[0147]

[0148] Table 8 Drop-off ddPCR detection results of CEBPA gene Q305dup mutant samples at different concentrations

[0149]

[0150] Table 9 Drop-off ddPCR detection results of CEBPA gene E309dup mutant samples at different concentrations

[0151]

[0152]

[0153] Table 10 Drop-off ddPCR detection results of CEBPA gene E309_T310insK mutant samples at different concentrations

[0154]

[0155] Table 11 Drop-off ddPCR detection results of CEBPA gene Q312dup mutant samples at different concentrations

[0156]

[0157]

[0158] Table 12 Drop-off ddPCR detection results of CEBPA gene K313dup mutant samples at different concentrations

[0159]

[0160] Table 13 Drop-off ddPCR detection results of CEBPA gene K313del mutant samples at different concentrations

[0161]

[0162] Table 14 Drop-off ddPCR detection results of CEBPA gene K313_V314insE mutant samples at different concentrations

[0163]

[0164]

[0165] Table 15 Drop-off ddPCR detection results of CEBPA gene E316_L317insR mutant samples at different concentrations

[0166]

[0167] Table 16 Drop-off ddPCR detection results of CEBPA gene L317dup mutant samples at different concentrations

[0168]

[0169] like Figure 23-32 As shown, the theoretical value and the actual detection value exhibit a good linear relationship. Therefore, the drop-off ddPCR method provided by the present invention can quantitatively detect common in-frame insertion / deletion mutations in the CEBPA gene, including K304_Q305insL, Q305dup, K313_V314insE, E316_L317insR, L317dup, E309dup, E309_T310insK, Q312dup, K313dup, and K313del mutations. Its lowest detection limit can reach 0.101%, and the detection is linear. The above description is merely a preferred embodiment of the present invention.

Claims

1. A primer probe set for detecting mutations in the bZIP region of the CEBPA gene, characterized in that: The primer-probe set includes a specific primer pair and probe set for detecting mutation sites in the bZIP region of the CEBPA gene; The nucleotide sequences of the specific primer pairs are shown in SEQ ID NO. 1-2; The nucleotide sequences of the probe groups are shown in SEQ ID NOs. 3 to 6 or SEQ ID NOs. 7 to 10.

2. The primer probe set according to claim 1, characterized in that The 5' end of the probe in the probe group is connected to a fluorescent group, and the 3' end is connected to a quenching group.

3. The primer probe set according to claim 2, characterized in that The fluorescent group is one or more of FAM, VIC, HEX, TRT, CY3, CY5, ROX and JOE, and the quenching group is one or more of TAMRA, DABCYL, MGB, BHQ-1, BHQ-2 and BHQ-3.

4. The primer probe set according to claim 1, characterized in that The mutations in the bZIP region of the CEBPA gene are in-frame insertion / deletion mutations including K304_Q305insL, Q305dup, K313_V314insE, E316_L317insR, L317dup, E309dup, E309_T310insK, Q312dup, K313dup, and K313del.

5. A kit for detecting mutations in the bZIP region of the CEBPA gene for non-diagnostic purposes, characterized in that: The method comprises the primer-probe set according to any one of claims 1 to 4.

6. The kit according to claim 5, characterized in that A CEBPA mutant plasmid was included as a positive control and a wild-type plasmid was included as a negative control.

7. A drop-off ddPCR method for quantitatively detecting mutations in the bZIP region of the CEBPA gene for non-diagnostic purposes, characterized in that: The following steps are involved: (1) Extracting DNA from the sample to be tested; (2) mixing the DNA of the sample to be tested with the components of the kit described in claims 5 to 6 to obtain a ddPCR reaction mixture; (3) After mixing the ddPCR reaction mixture with the oil phase, the resulting mixture is prepared into microreaction droplets using a microdroplet preparation instrument; (4) After the micro-reaction droplets are introduced into the chip, a PCR amplification reaction is performed, and signals of the micro-droplets in the chip after the PCR amplification reaction are collected to calculate the allele mutation frequency of the CEBPA gene in the sample to be tested.

8. The drop-off ddPCR method according to claim 7, characterized in that The sample to be tested in step (1) is the patient's peripheral blood, bone marrow tissue or tumor tissue.

9. The drop-off ddPCR method according to claim 7, characterized in that The ddPCR reaction mixture in step (2) includes the following components: 10-20 μl of 2×dPCR reaction buffer, 0.4-0.6 μl of SNUPP DNA polymerase, 22-26 pmol each of the specific primers shown in SEQ ID NOs. 1-2, 2-4 pmol each of the probes shown in SEQ ID NOs. 3-6 or SEQ ID NOs. 7-10, 40-500 ng of the test sample DNA, and nuclease-free water to make up to 30 μl.

10. The drop-off ddPCR method according to claim 7, characterized in that The PCR amplification reaction procedure in step (4) is: 50°C for 5 min, 95°C for 5 min, 96°C for 15 s, 60-65°C for 30 s, 40-50 cycles, and finally maintained at 25°C.

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