Primer probe set, kit and detection method for detecting biallelic CEBPA gene mutations

Abstract

The present invention relates to the field of biological detection technology, and discloses a primer probe set, a kit, and a detection method for detecting biallelic CEBPA gene mutations. The primers are used for full-length amplification of the CEBPA gene coding region, and include an upstream primer and a downstream primer, the upstream primer having a nucleotide sequence as shown in SEQ ID No: 1, and the downstream primer having a nucleotide sequence as shown in SEQ ID No: 2; the probe hybridizes with the CEBPA gene sequence and introduces a single mutation on either side of the N-terminus of the CEBPA gene where a single base mutation occurs. The detection method provided by the present invention has high specificity and accuracy. The primers, probes, and detection method provided by the present invention can effectively detect whether two mutations in the CEBPA gene are biallelic mutations, providing strong technical support and reliable detection means for further determining the prognosis of AML patients.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a primer probe set, a kit and a detection method for detecting biallelic CEBPA gene mutations. Background Art

[0002] Alterations in cell proliferation, differentiation, and apoptosis pathways caused by gene mutations underlie the pathogenesis of acute myeloid leukemia (AML). Recently, several molecular markers with significant prognostic value have been identified in normal karyotype AML. Among them, the myeloid transcription factor CCA AT-enhancer binding protein-alpha (CEBPA) is exclusively expressed in myeloid cells within the hematopoietic system. It is a crucial transcription factor in the proliferation and differentiation of myeloid progenitor cells, inducing granulocyte differentiation and inhibiting proliferation. This transcription factor consists of 358 amino acids and is encoded by the gene CEBPA. The CEBPA gene is located on chromosome 19 and is 3318 bp long, with no introns in the sequence. The cDNA is 1182 bp long. Its structure comprises an N-terminal transcriptionally active region, a DNA binding region, and a C-terminal leucine-rich dimerization domain. Under normal circumstances, the full-length 42kD protein predominates, with a small amount of the 30kD protein also present. Changes in the ratio of the two can alter its function. In the newly revised WHO leukemia classification criteria, detection of CEBPA gene mutations has become an important diagnostic, stratification, and prognostic indicator for AML and may become a new therapeutic target.

[0003] Mutations in the CEBPA gene occur in approximately 5% to 15% of AML patients, predominantly in the M1 and M2 subtypes. Two main types of mutations exist: N-terminal frameshift mutations, which prematurely terminate translation of the normal p42 protein and initiate translation from an alternative start codon into a truncated p30 protein. C-terminal insertions, deletions, duplications, and substitutions, typically in-frame mutations, result in proteins lacking DNA binding and homodimerization activity. Patients can harbor either monoallelic or biallelic mutations (i.e., two mutations located on different chromosomes within the CEBPA gene, typically one at the N-terminal site and the other at the C-terminal site). Notably, biallelic mutations are considered a marker of favorable prognosis. The 2016 World Health Organization classification of myeloid neoplasms lists AML with biallelic CEBPA mutations as a separate entity to guide the diagnosis and treatment of this type of leukemia. Therefore, CEBPA biallelic mutation detection is of great value in the diagnosis and treatment of AML. Accurate test results not only help to identify the disease early, but also provide an indispensable decision-making basis for the formulation of subsequent personalized treatment plans.

[0004] Currently, CEBPA detection is primarily based on genetic testing technologies. Through methods such as PCR and sequencing, changes in the CEBPA gene sequence, including point mutations, insertions, and deletions, can be detected. However, in some AML patients, the CEBPA gene often exhibits two mutations. Existing detection methods, such as PCR and sequencing, can only confirm the presence of two mutations in the gene and cannot clearly determine whether these two mutations are biallelic. These limitations of existing detection methods have, to a certain extent, hindered disease detection, precision treatment, and prognosis assessment in AML patients. Summary of the Invention

[0005] In view of this, and addressing the deficiencies of the above-mentioned prior art, the present invention aims to provide primers, probes, a kit, and a detection method for detecting biallelic CEBPA gene mutations. The primers provided by the present invention can directly amplify the full length of the CEBPA coding region using a two-step PCR method, thereby improving the specificity of amplification and reducing the probability of mismatches. By artificially introducing mismatches into the probe, the disadvantage of T7 endonuclease I's poor ability to recognize single-base mutations can be circumvented. The detection method provided by the present invention has high specificity and accuracy. The primers, probes, and detection method provided by the present invention can effectively detect whether the two mutations in the CEBPA gene are biallelic mutations, providing strong technical support and a reliable detection method for further determining the prognosis of AML patients.

[0006] To solve the above technical problems, this application provides the following technical solutions.

[0007] In a first aspect, the present invention provides primers and probes for detecting biallelic CEBPA gene mutations. The primers are used for full-length amplification of the CEBPA gene coding region and include an upstream primer and a downstream primer. The upstream primer has a nucleotide sequence as shown in SEQ ID No: 1, and the downstream primer has a nucleotide sequence as shown in SEQ ID No: 2. The probe hybridizes with the CEBPA gene sequence and introduces single mutations on both sides of the N-terminus of the CEBPA gene where a single base mutation occurs.

[0008] Preferably, the probe has a nucleotide sequence as shown in SEQ ID No: 3.

[0009] In a second aspect, the present invention provides a kit for detecting biallelic CEBPA gene mutations, the kit comprising the above-mentioned primers, the above-mentioned probe and T7 endonuclease I.

[0010] In a third aspect, the present invention provides a method for detecting biallelic CEBPA gene mutations, comprising the following steps:

[0011] Step 1: extract the DNA of the sample to be tested, and use the DNA of the sample to be tested as a template and the above primers to perform a PCR amplification reaction to obtain a PCR product of the sample to be tested;

[0012] Step 2: extracting the plasmid solution of the probe, and using the probe plasmid solution as a template and the primers as described above to perform PCR amplification reaction to obtain a probe PCR product;

[0013] Step 3: Mix equal volumes of the test sample PCR product from step 1 and the probe PCR product from step 2, then denature and anneal. After denaturation and annealing, add T7 endonuclease I for enzyme digestion. The digestion products are subjected to agarose gel electrophoresis. Based on the electrophoresis results, determine whether the two mutations in the test sample are biallelic mutations.

[0014] Furthermore, the method for determining whether the two mutations in the sample to be tested are biallelic mutations in step 3 is as follows: observing the DNA bands in the 500bp-1000bp length segment in the electrophoresis diagram; if a bright DNA band can be clearly observed in the 500bp-1000bp length segment, the two mutations in the sample to be tested are located on the same chromosome; if no bright DNA band is observed in the 500bp-1000bp length segment, the two mutations in the sample to be tested are located on different chromosomes.

[0015] Furthermore, the PCR amplification reaction procedures of step 1 and step 2 include the following steps: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing and extension at 72°C for 90 seconds, 35 cycles; extension at 72°C for 7 minutes; and storage at 4°C.

[0016] Furthermore, the denaturation annealing procedure in step 3 includes the following steps: pre-denaturation at 95°C for 5 minutes; cooling from 95°C to 85°C at a rate of 0.2°C / s; cooling from 85°C to 25°C at a rate of 0.1°C / s; and storage at 4°C.

[0017] Furthermore, the enzymatic digestion reaction in step 3 includes the following steps: enzymatic incubation at 37° C. for 40 min; terminating the enzymatic digestion reaction at 85° C. for 15 min; and storing at 4° C.

[0018] Furthermore, in step 1, the target fragment amplification reaction system contains the following components per 20 μL: 10 μL of 2× reaction solution, 4 μL of 5× PCR enhancer, 2 μL of ultrapure water, 1 μL each of upstream and downstream primers with a concentration of 10 μM, and 2 μL of DNA template with a concentration of 30-50 ng / μL.

[0019] Furthermore, the target fragment amplification reaction system in step 2 contains the following components per 20 μL: 10 μL of 2× reaction solution, 4 μL of 5× PCR enhancer, 2 μL of ultrapure water, 1 μL each of the upstream and downstream primers with a concentration of 10 μM, and 1 μL of the upstream and downstream primers with a concentration of 10 μM. 6 / μL of probe plasmid liquid template 2μL.

[0020] Compared with the prior art, the present invention is described in detail as follows:

[0021] The detection method provided by the present invention is based on the premise that two mutations are known in the CEBPA gene. The present invention mainly utilizes the property of T7 endonuclease I (T7EI) to recognize and cleave incompletely paired DNA double strands. Combined with agarose gel electrophoresis technology, the DNA fragments treated with T7EI enzyme are subjected to electrophoresis analysis. Based on the electrophoresis results, it is inferred whether the two mutations are biallelic mutations. The principle is as follows. Figure 1 As shown, the two mutations are located on the same chromosome (the first double mutation type) and on different chromosomes (the second double mutation type). The lengths of the nucleic acid fragments formed after cleavage by T7EI enzyme are different. Therefore, it can be judged whether the two mutations are located on the same chromosome or different chromosomes based on the different lengths of nucleic acid fragments represented on the electrophoresis graph.

[0022] In early experiments, the inventors discovered that T7E1's recognition of single-base mutations was unstable, and literature reported that T7E1 was highly sensitive to consecutive multi-base mutations. Therefore, the inventors designed and prepared a probe targeting common point mutations in CEBPA. This probe provides an auxiliary judgment tool for detecting specific single-base mutations and consecutive base mismatches of two or more bases. By artificially introducing mismatches in the probe, the inventors circumvented T7E1's poor recognition of single-base mutations.

[0023] The specific primers designed in the present invention can be used to directly amplify the full-length CEBPA coding region using a two-step PCR method (i.e., annealing and extension are performed simultaneously at the same temperature). Compared with the three-step PCR method, this method eliminates one heating and cooling process, increases the reaction speed, enhances the specificity of amplification, and reduces the probability of mismatches. This is of great significance for accurately determining the type of CEBPA double mutation.

[0024] The detection method provided by the present invention has high specificity and accuracy, and can effectively detect whether the two mutations in the CEBPA gene are biallelic mutations, providing strong support and guarantee for the design and implementation of subsequent personalized medical treatment for patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the principle of CEBPA biallelic mutation detection of the present invention;

[0026] Figure 2 The electrophoresis pattern of the sample DNA amplified using the optimal primer pair in Example 1;

[0027] Figure 3 This is the electrophoresis pattern of the fifth step in Example 3;

[0028] Figure 4 This is the electrophoresis pattern of the ninth step in Example 3;

[0029] Figure 5 is the electrophoresis pattern of Example 5;

[0030] Figure 6 This is the electrophoresis pattern of Example 6. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention is further described in detail below with reference to specific embodiments.

[0032] Example 1: Primers

[0033] The present invention provides a primer capable of full-length amplification of the CEBPA coding region in a two-step method, the design of which is as follows:

[0034] First, the full-length CEBPA coding region sequence was retrieved from the NCBI database (https: / / www.ncbi.nlm.nih.gov). Specific primers were designed using Primer Explorer software (version 5.0). Among the multiple pairs of synthesized primers, the optimal primer pair was screened using the following steps.

[0035] The optimal primer pair screening method includes the following steps:

[0036] Step 1: Extract the sample DNA to be amplified and adjust the DNA concentration to 30-50 ng / μL to obtain a sample DNA template; the sample DNA can be extracted using a commercial detection kit or a fully automatic nucleic acid extraction instrument.

[0037] Step 2: Dissolve the designed upstream primer and downstream primer into 10 μM working solution and mix them in equal volumes to prepare a primer mixture.

[0038] Step 3. Prepare the PCR reaction system: Add 10 μL of 2× reaction solution, 4 μL of 5× PCR enhancer, 2 μL of ultrapure water, 2 μL of primer mix, and 2 μL of sample DNA template to a transparent 200 μL reaction tube. Mix and centrifuge briefly. Both the 2× reaction solution and the 5× PCR enhancer are commercially available products. The main components of the 2× reaction solution include Taq DNA polymerase, dNTPs, MgCl2, and optimized DreamTaq buffer. The main components of the 5× PCR enhancer include HLTaq DNA polymerase, 10× Balb PCR buffer, and dNTP mixture.

[0039] Step 4, PCR amplification: Place the reaction tube into the PCR amplification instrument. The PCR amplification reaction conditions are as follows: (1) pre-denaturation at 95°C for 5 min; (2) denaturation at 95°C for 30 s, annealing and extension at 72°C for 90 s, 35 cycles; (3) extension at 72°C for 7 min; (4) storage at 4°C.

[0040] Step 5: Agarose gel electrophoresis analysis of amplification effect: Prepare 2% agarose gel and perform DNA electrophoresis.

[0041] Through the above method, the present invention screened out the optimal primer pairs as follows:

[0042] Upstream primer: CEBPAfull-F (sequence shown in SEQ ID No: 1);

[0043] Downstream primer: CEBPAfull-R (sequence shown in SEQ ID No: 2).

[0044] The electrophoresis pattern after DNA amplification using the above optimal primer pair is shown in Figure 2 As shown, Figure 2 Lane 1 is DL1000bp DNA marker; Lane 14 is DL5000bp DNA marker; Lanes 2-13 are 12 randomly selected samples, of which Lane 12 is a blank control (unamplified sample). DL1000bp DNA marker consists of DNA fragments of 1000bp, 700bp, 500bp, 400bp, 300bp, 200bp, and 100bp, for a total of 7 bands; DL5000bp DNA marker consists of DNA fragments of 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, and 100bp, for a total of 9 bands. Figure 2The bands in lanes 2-11 and 13 show that the PCR products are of the same size, indicating that the optimal primer pair provided by the present invention can extend the full length of the CEBPA gene coding region without any non-specific bands.

[0045] Example 2: Probe

[0046] The present invention provides a probe for detecting whether a CEBPA point mutation is biallelic. This probe introduces single mutations on either side of the N-terminus of the CEBPA gene where a single base mutation occurs, thereby circumventing the shortcoming of T7EI's poor recognition ability for single base mutations. The design concept is as follows:

[0047] A search of the COSMIC database (https: / / cancer.sanger.ac.uk / ) for common mutations in the CEBPA coding region revealed that single base changes were predominant at the N-terminus, while two or three base changes were predominant at the C-terminus. Based on the characteristics of the enzyme used in the experiment, single mutations were introduced on either side of the N-terminus where single base mutations frequently occur (e.g., if the original sequence is AACTCCG, the mutated sequence is AACGGCCG, and the artificially introduced mutation sequence is AATTACG). This artificially synthesized mutant sequence, named CEBPA-mut (sequence shown in SEQ ID No: 13), was used as an auxiliary detection tool. The desired mutant sequence was submitted to a biotechnology company for synthesis, which ultimately provided cloning strains and plasmid powder containing the target fragment. Artificial gene sequence synthesis can be performed by consulting a professional biotechnology company.

[0048] Example 3: Preparation of samples with two CEBPA mutations located on the same chromosome

[0049] Since sufficient samples with double mutations on the same DNA strand were not found, in order to test the feasibility of the technical method, the test samples needed to be prepared according to the following method.

[0050] Step 1: Combine laboratory testing of existing specimens and select samples found to have the c.937-939dup single mutation by NGS or Sanger sequencing. Extract sample DNA and adjust the DNA concentration to 30-50 ng / μL to obtain the sample DNA template.

[0051] Step 2: Dissolve the upstream and downstream primers shown in Table 1 below into 10 μM working solutions, mix them in equal volumes to prepare a primer mix. The labels of the primer mix correspond to the reaction tube numbers.

[0052] Step 3: To eight transparent 200μL reaction tubes, labeled 1, 2, 3, 4, 5, 6, 7, and 8, add 10μL of 2× reaction solution, 4μL of 5× PCR enhancer, 2μL of ultrapure water, 2μL of primer mix, and 2μL of sample DNA template. Mix and centrifuge briefly. The primer mix added to each tube corresponds to the reaction tube number.

[0053] Table 1. Amplification primers

[0054]

[0055]

[0056] Remark:

[0057] The sequence of c.58CT-F is shown in SEQ ID No: 3;

[0058] The sequence of c.58CT-R is shown in SEQ ID No: 4;

[0059] The sequence of c.68del-F is shown in SEQ ID No: 5;

[0060] The sequence of c.68del-R is shown in SEQ ID No: 6;

[0061] The sequence of c.179dup-F is shown in SEQ ID No: 7;

[0062] The sequence of c.179dup-R is shown in SEQ ID No: 8;

[0063] The sequence of c.247del-F is shown in SEQ ID No: 9;

[0064] The sequence of c.247del-R is shown in SEQ ID No: 10;

[0065] The sequence of c.937-939-F is shown in SEQ ID No: 11;

[0066] The sequence of c.937-939-R is shown in SEQ ID No: 12.

[0067] Step 4: Place each reaction tube into a PCR amplification instrument. The PCR amplification reaction conditions are as follows: (1) pre-denaturation at 95°C for 5 minutes; (2) denaturation at 95°C for 30 seconds; (3) annealing and extension at 72°C for 90 seconds; (4) (2)-(3) reaction cycles 35 times; (5) extension at 72°C for 7 minutes; (6) storage at 4°C.

[0068] Step 5: Prepare 2% agarose gel and perform DNA electrophoresis analysis on the amplified products in each reaction tube. The results are as follows: Figure 3 shown. Figure 3 Lanes 1 and 10 are DL1000 DNA markers; lanes 2-9 are amplified fragments. The size of the PCR products is consistent with expectations, and there are no nonspecific bands.

[0069] Step 6: Take four transparent 200 μL reaction tubes, mark them as 9, 10, 11, and 12, add 10 μL of 2X reaction solution, 4 μL of 5X PCR enhancer, 2 μL of ultrapure water, and 2 μL of primer mixture (CEBPAfull-F and CEBPAfull-R) and mix them.

[0070] Step 7: Mix equal volumes of reaction solutions No. 1 and No. 2 after amplification in the fourth step to obtain mixed solution 1, mix equal volumes of reaction solutions No. 3 and No. 4 to obtain mixed solution 2, mix equal volumes of reaction solutions No. 5 and No. 6 to obtain mixed solution 3, and mix equal volumes of reaction solutions No. 7 and No. 8 to obtain mixed solution 4; add 2 μL of mixed solution 1 to reaction tube No. 9, 2 μL of mixed solution 2 to reaction tube No. 10, 2 μL of mixed solution 3 to reaction tube No. 11, and 2 μL of mixed solution 4 to reaction tube No. 12, so that the final volume of the reaction solution in each tube is 20 ul, and centrifuge instantly after mixing.

[0071] Step 8: Place reaction tubes No. 9-12 into the PCR amplification instrument. The PCR amplification reaction conditions are as follows: (1) pre-denaturation at 95°C for 5 minutes; (2) denaturation at 95°C for 30 seconds; (3) annealing and extension at 72°C for 90 seconds; (4) (2)-(3) reaction cycles 35 times; (5) extension at 72°C for 7 minutes; (6) storage at 4°C.

[0072] Step 9: Prepare 2% agarose gel and perform DNA electrophoresis analysis on the amplified products in reaction tubes 9-12. The results are as follows: Figure 4 shown. Figure 4 Lane 5 in the middle is the DL1000 DNA marker; lanes 1-4 are the amplified fragments from reaction tubes 9, 10, 11, and 12. The size of the PCR products is consistent with expectations, and there are no nonspecific bands.

[0073] The reaction solution in reaction tube No. 9 is the PCR product of the sample in which the c.58CT and c.937-939dup mutations are located on the same chromosome.

[0074] The reaction solution in reaction tube No. 10 is the PCR product of the sample in which the c.68del and c.937-939dup mutations are located on the same chromosome.

[0075] The reaction solution in reaction tube No. 11 is the PCR product of the sample in which the c.179dup and c.937-939dup mutations are located on the same chromosome.

[0076] The reaction solution in reaction tube No. 12 is the PCR product of the sample in which the c.247del and c.937-939dup mutations are located on the same chromosome.

[0077] Example 4: Detection of Bi-allelic Mutations in CEBPA

[0078] See also Figure 1 The principle of the method for detecting CEBPA biallelic mutations provided by the present invention is as follows: First, the samples to be tested are amplified to obtain corresponding PCR products, which are then randomly matched into double-stranded DNA by high-temperature denaturation and slow cooling and annealing. Next, the properties of the T7E1 enzyme are utilized to identify and cleave incompletely paired DNA. Depending on the location of the mismatch, the PCR products are cut into fragments of different lengths. Finally, the band sizes on agarose gel electrophoresis are used to determine whether the two mutations are on the same DNA strand. (Note: Figure 1 Lane M represents marker, and lane CT represents blank control).

[0079] The above detection method specifically comprises the following steps:

[0080] Step 1: Prepare the annealing reaction system. Take a transparent 200 μL reaction tube and add 2 μL of 10×T7EI reaction buffer, 10 μL of enzyme-free water, 3.5 μL of CEBPA-mut PCR product, and 3.5 μL of the sample PCR product to make the liquid volume in the reaction tube 19 μL. Mix and centrifuge briefly.

[0081] Step 2: Perform annealing reaction. Place the reaction tube into the PCR amplification instrument and set the reaction conditions as follows: (1) pre-denaturation at 95°C for 5 min; (2) Annealing 1: cooling from 95°C to 85°C at a rate of 0.2°C / s; (3) Annealing 2: cooling from 85°C to 25°C at a rate of 0.1°C / s; (4) storage at 4°C.

[0082] Step 3: Take the system after the second step annealing reaction out of the PCR instrument, add 1μL T7EI to the reaction tube, put the reaction tube back into the PCR amplification instrument, and set the reaction conditions as follows: (1) incubate with enzyme digestion at 37℃ for 40min; (2) terminate the enzyme digestion reaction at 85℃ for 15min; (3) store at 4℃.

[0083] Step 4: Prepare 2% agarose, perform agarose gel electrophoresis on the enzyme-digested products in the reaction tube, take photos and observe, and analyze the enzyme-digested effects.

[0084] Among them, 10×T7EI reaction buffer is a commercially available product; the CEBPA-mut PCR product is obtained by extracting the probe plasmid solution of Example 2 and using the plasmid solution as the sample DNA template, and performing PCR amplification according to the PCR amplification method in the optimal primer pair screening method provided in Example 1; the PCR product of the sample to be tested is obtained by PCR amplification according to the PCR amplification method in the optimal primer pair screening method provided in Example 1.

[0085] The extraction method of the above-mentioned probe plasmid solution is as follows:

[0086] (1) Take a 15 ml centrifuge tube and inoculate the bacterial cells synthesized by the biological company in Example 2 into 5 ml of LB liquid medium containing 1‰ ampicillin, and culture on a shaking platform for 14 h (37°C, 220 rpm);

[0087] (2) Using a commercial plasmid extraction kit, the CEBPA-mut plasmid was extracted from the above culture medium and diluted to a concentration of 10 6 / μL.

[0088] Example 5: Verification of the detection method of Example 4 1

[0089] Based on the database and laboratory test data, two double mutation samples of different mutation types were selected for testing (c.58CT and c.937-939dup; c.68del and c.937-939dup). It is known that the reaction solution in reaction tube 9 in Example 3 is the PCR product of a sample in which the c.58CT and c.937-939dup mutations are located on the same chromosome; the reaction solution in reaction tube 10 is the PCR product of a sample in which the c.68del and c.937-939dup mutations are located on the same chromosome.

[0090] Test results are shown in Figure 5 As shown, lanes 1, 9, and 17 are DL1000 DNA markers, lanes 8 and 16 are full-length gene fragments that have not been digested by enzymes, lanes 2, 4, 6, 10, 11, and 14 are electrophoretic images of c.58CT and c.937-939dup mutations on different chromosomes; lanes 3, 5, 7, 12, 13, and 15 are electrophoretic images of c.58CT and c.937-939dup mutations on the same chromosome; lanes 18, 26, and 34 are DL1000 DNA markers, lanes 8 and 16 are full-length gene fragments that have not been digested by enzymes, lanes 2, 4, 6, 10, 11, and 14 are electrophoretic images of c.58CT and c.937-939dup mutations on different chromosomes; lanes 3, 5, 7, 12, 13, and 15 are electrophoretic images of c.58CT and c.937-939dup mutations on the same chromosome. marker, lanes 25 and 33 are the full-length gene fragments that have not been digested by enzymes, lanes 19, 21, 23, 27, 29, and 31 are the electrophoretic patterns of c.68del and c.937-939dup mutations on different chromosomes; lanes 20, 22, 24, 28, 30, and 32 are the electrophoretic patterns of c.68del and c.937-939dup mutations on the same chromosome.

[0091] Example 6: Verification 2 of the detection method of Example 4

[0092] Based on the database and laboratory test data, two double mutation samples of different mutation types were selected for testing (c.179dup and c.937-939dup; c.247del and c.937-939dup). It is known that the reaction solution in reaction tube No. 11 in Example 3 is the PCR product of a sample in which the c.179dup and c.937-939dup mutations are located on the same chromosome; the reaction solution in reaction tube No. 12 is the PCR product of a sample in which the c.247del and c.937-939dup mutations are located on the same chromosome.

[0093] Test results are shown in Figure 6 As shown, lanes 1, 9, and 17 are DL1000 DNA markers, lanes 2 and 10 are full-length gene fragments that have not been digested by enzymes, lanes 3, 5, 7, 11, 13, and 15 are electrophoretic images of c.179dup and c.937-939dup mutations on different chromosomes; lanes 4, 6, 8, 12, 14, and 16 are electrophoretic images of c.179dup and c.937-939dup mutations on the same chromosome; lanes 18, 26, and 34 ... marker, lanes 19 and 27 are the full-length gene fragments that have not been digested by enzymes, lanes 20, 22, 24, 28, 30, and 32 are the electrophoretic patterns of c.247del and c.937-939dup mutations on different chromosomes; lanes 21, 23, 25, 29, 31, and 33 are the electrophoretic patterns of c.247del and c.937-939dup mutations on the same chromosome.

[0094] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A kit for detecting biallelic CEBPA gene mutations, characterized by: The kit includes T7 endonuclease I and primers and probes for detecting biallelic CEBPA gene mutations. The primers are used to amplify the full-length coding region of the CEBPA gene and include an upstream primer and a downstream primer. The upstream primer has a nucleotide sequence as shown in SEQ ID No: 1, and the downstream primer has a nucleotide sequence as shown in SEQ ID No:

2. The probe hybridizes with the CEBPA gene sequence and introduces single mutations on both sides of the N-terminus of the CEBPA gene where a single base mutation occurs.

2. The kit according to claim 1, wherein: The probe has a nucleotide sequence as shown in SEQ ID No:

13.

3. The kit according to claim 1, wherein: The procedure for PCR amplification reaction using the kit includes the following steps: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing and extension at 72°C for 90 seconds, 35 cycles; extension at 72°C for 7 minutes; and storage at 4°C.

4. The kit according to claim 3, wherein: The denaturation and annealing procedure of the PCR amplification reaction product includes the following steps: pre-denaturation at 95°C for 5 minutes; cooling from 95°C to 85°C at a rate of 0.2°C / s; cooling from 85°C to 25°C at a rate of 0.1°C / s; and storage at 4°C.

5. The kit according to claim 1, wherein: The enzyme digestion reaction using the kit includes the following steps: Incubate the enzyme digestion at 37°C for 40 min; terminate the enzyme digestion reaction at 85°C for 15 min; and store at 4°C.

6. The kit according to claim 3, wherein: The PCR amplification reaction system contained the following components per 20 μL: 10 μL of 2× reaction solution, 4 μL of 5× PCR enhancer, 2 μL of ultrapure water, 1 μL each of upstream and downstream primers with a concentration of 10 μM, and 2 μL of DNA template with a concentration of 30-50 ng / μL.

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