Primer probe group, kit and detection method for detecting double-allelic CEBPA gene mutation

Abstract

The invention relates to the technical field of biological detection, and discloses a primer probe group, a kit and a detection method for detecting double-allelic CEBPA gene mutation, the primer is used for full-length amplification of a CEBPA gene coding region and comprises 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 downstream primer has a nucleotide sequence as shown in SEQ ID No: 2; the probe is hybridized with a CEBPA gene sequence, and single mutation is introduced to the two sides of the N end, with single base mutation, of the CEBPA gene respectively. The detection method provided by the invention has high specificity and accuracy, and by adopting the primer, the probe and the detection method provided by the invention, whether two mutations of the CEBPA gene belong to diallele mutation can be effectively detected, and a powerful technical support and a reliable detection means are provided for further judging the prognosis condition of an AML patient.

Description

Technical Field

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

[0002] Changes in cell proliferation, differentiation and apoptosis pathways caused by gene mutations are the pathogenesis basis of acute myeloid leukemia (AML). Recently, a variety of molecular markers with important prognostic value have been identified in AML with normal karyotype. Among them, the myeloid transcription factor CCAAT / enhancer-binding protein alpha (CEBPA) is only expressed in myeloid cells in the hematopoietic system. It is an important transcription factor in the process of proliferation and differentiation of myeloid progenitor cells, and has the effects of inducing granulocytic differentiation and inhibiting proliferation. This transcription factor contains 358 amino acids and is encoded by the gene CEBPA. The gene CEBPA is located on chromosome 19, with a full length of 3318 bp, no introns in the sequence, and a cDNA length of 1182 bp. Its structure includes: an N-terminal transcriptional activation region, a DNA-binding region, and a C-terminal leucine-rich dimerization functional region. Under normal circumstances, the full-length 42 kD protein is the main form, and there is also a small amount of 30 kD protein. The change in the ratio between the two will lead to the change of its function. In the newly revised WHO leukemia classification standard, the detection of CEBPA gene mutations has become an important diagnostic, stratification and prognostic reference index for AML, and may become a new therapeutic target.

[0003] CEBPA gene mutations are found in about 5% - 15% of AML patients, mostly in subtypes M1 and M2. Its mutations mainly have two forms: one is a frameshift mutation at the N-terminus, which leads to premature termination of the translation of the normal p42 protein and translation of a truncated p30 protein starting from another start codon; the other is base insertion, deletion, duplication and substitution at the C-terminus, usually an in-frame mutation, and the resulting protein lacks DNA-binding and homodimer activity. Patients can have either single-allele gene mutations or biallelic gene mutations (that is, two mutations on different chromosomes in the allelic CEBPA gene, generally one at the N-terminal site and the other at the C-terminal site). It is worth noting that biallelic gene mutations are considered a sign of good prognosis. In the 2016 World Health Organization classification system of myeloid neoplasms, AML with CEBPA biallelic gene mutations is listed as an independent disease entity to guide the diagnosis and treatment of such leukemia. Therefore, the detection of CEBPA biallelic gene mutations has important value in the diagnosis and treatment of AML. Accurate detection results not only help in the early identification of the disease, but also provide an indispensable decision-making basis for the formulation of subsequent personalized treatment plans.

[0004] Currently, the detection of CEBPA is mainly based on gene detection technology. Through methods such as PCR and sequencing, changes in the CEBPA gene sequence can be detected, including point mutations, insertions, deletions, etc. However, in some AML patients, the CEBPA gene often has two mutations, and existing detection methods such as PCR and sequencing can only determine that there are two mutations in the gene, and cannot clarify whether these two mutations belong to biallelic gene mutations. The limitations of existing detection methods have, to a certain extent, hindered the disease detection, precise treatment, and prognosis evaluation of AML patients. Summary of the Invention

[0005] In view of this, aiming at the above deficiencies of the existing technology, the purpose of the present invention is to provide a primer, probe, kit, and detection method for detecting biallelic CEBPA gene mutations. The primer provided by the present invention can directly amplify the full length of the CEBPA coding region by two-step PCR, improving the specificity of amplification and reducing the probability of mismatch occurrence; by artificially introducing mismatches through the probe, the disadvantage that T7 endonuclease I has poor recognition ability for single-base mutations can be avoided; the detection method provided by the present invention has high specificity and accuracy. Using the primer, probe, and detection method provided by the present invention can effectively detect whether the two mutations of the CEBPA gene belong to biallelic gene mutations, providing strong technical support and reliable detection means for further judging the prognosis of AML patients.

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

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

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

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

[0010] In the third aspect, the present invention provides a method for detecting biallelic CEBPA gene mutations, which includes 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 to perform a PCR amplification reaction with the primers described in claim 1 to obtain the PCR product of the sample to be tested;

[0012] Step 2: Extract the plasmid solution of the probe described in claim 1 or 2, and use the probe plasmid solution as a template to perform a PCR amplification reaction with the primers described in claim 1 to obtain the PCR product of the probe;

[0013] Step 3: Mix the PCR product of the sample to be tested in Step 1 and the PCR product of the probe in Step 2 in equal volumes and perform denaturation and annealing. After the denaturation and annealing are completed, add T7 endonuclease I for enzymatic digestion reaction. Electrophorese the enzymatic digestion product on an agarose gel, and judge whether the two mutations of the sample to be tested belong to biallelic gene mutations according to the electrophoresis results.

[0014] Further, the method for judging whether the two mutations of the sample to be tested belong to biallelic gene mutations in Step 3 is as follows: Observe the DNA band in the length range of 500bp - 1000bp in the electrophoresis pattern. If the band length < 1000bp, the two mutations of the sample to be tested are located on the same chromosome; if the band length ≥ 1000bp, the two mutations of the sample to be tested are located on different chromosomes.

[0015] Further, the PCR amplification reaction procedures in Step 1 and Step 2 include the following steps: Pre-denature at 95°C for 5 min; Denature at 95°C for 30 s, anneal and extend at 72°C for 90 s, cycle 35 times; Extend at 72°C for 7 min; Store at 4°C.

[0016] Further, the denaturation and annealing procedure in Step 3 includes the following steps: Pre-denature at 95°C for 5 min; Cool from 95°C to 85°C at a rate of 0.2°C / s; Cool from 85°C to 25°C at a rate of 0.1°C / s; Store at 4°C.

[0017] Further, the enzymatic digestion reaction in Step 3 includes the following steps: Incubate at 37°C for enzymatic digestion for 40 min; Terminate the enzymatic digestion reaction at 85°C for 15 min; Store at 4°C.

[0018] Further, each 20 μL of the target fragment amplification reaction system in Step 1 contains the following components: 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] Further, each 20 μL of the target fragment amplification reaction system in Step 2 contains the following components: 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 a concentration of 106 2 μL of the probe plasmid solution template at / μL.

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

[0021] The detection method provided by the present invention is carried out on the premise that two mutations of the CEBPA gene are known. The present invention mainly utilizes the characteristic that T7 endonuclease I (T7EI) can recognize and digest incompletely paired DNA double strands, combined with agarose gel electrophoresis technology, to perform electrophoresis analysis on the DNA fragments after being treated with T7EI enzyme, and infer whether the two mutations belong to biallelic mutations according to the electrophoresis results. The principle is as Figure 1 shown. The lengths of the nucleic acid fragments formed after the two mutations located on the same chromosome (the first type of double mutation) and different chromosomes (the second type of double mutation) are sheared by T7EI enzyme are different, so it can be judged whether the two mutations are located on the same chromosome or different chromosomes according to the nucleic acid fragments with different lengths characterized on the electrophoresis map.

[0022] In the preliminary experiment, the inventors found that the recognition of single-base mutations by T7E1 is unstable, and there are literature reports that T7E1 shows excellent sensitivity to continuous multi-base mutations. Therefore, the inventors designed and prepared a probe for the common point mutations of CEBPA, which can provide an auxiliary judgment tool for detecting specific single-base mutations and base mismatches of two or more consecutive bases. By artificially introducing mismatches through the probe, the disadvantage of poor recognition ability of T7EI for single-base mutations is avoided.

[0023] The specific primers designed by the present invention can directly amplify the full length of the CEBPA coding region by two-step PCR (i.e., annealing and extension are carried out at the same temperature), compared with three-step PCR, it reduces one heating and cooling process, improves the reaction speed, enhances the specificity of amplification, reduces the probability of mismatch occurrence, and is of great significance for the accurate judgment of the CEBPA double mutation type.

[0024] The detection method provided by the present invention has high specificity and accuracy, and can effectively detect whether the two mutations of the CEBPA gene belong to 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 is a schematic diagram of the principle of CEBPA biallelic mutation detection of the present invention;

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

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

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

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

[0030] Figure 6 It is the electrophoresis pattern of Example 6. Detailed implementation manners

[0031] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below in conjunction with specific embodiments.

[0032] Example 1: Primers

[0033] The present invention provides a pair of primers for two-step full-length amplification of the CEBPA coding region, and the design idea is as follows:

[0034] First, retrieve the full-length sequence of the CEBPA coding region in the NCBI database (https: / / www.ncbi.nlm.nih.gov), design specific primers through primer design software (Primer Explorer software, version 5.0), and screen the optimal primer pair through the following steps among the synthesized multiple pairs of primers.

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

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

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

[0038] Step 3: Prepare a PCR reaction system: Take a transparent 200 μL reaction tube, add 10 μL of 2× reaction solution, 4 μL of 5×PCR enhancer, 2 μL of ultrapure water, 2 μL of primer mixture, and 2 μL of sample DNA template thereto, and 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 an optimized DreamTaq buffer, and the main components of the 5×PCR enhancer include HL Taq DNA Polymerase, 10×Balb PCR Buffer, dNTP Mixture, etc.

[0039] Step 4, PCR amplification: Place the reaction tube into a PCR amplifier, and the PCR amplification reaction conditions are as follows: (1) Pre-denature at 95°C for 5 min; (2) Denature at 95°C for 30 s, anneal and extend at 72°C for 90 s, for 35 cycles; (3) Extend at 72°C for 7 min; (4) Store at 4°C.

[0040] Step 5, Analyze the amplification effect by agarose gel electrophoresis: Prepare 2% concentration agarose for DNA electrophoresis.

[0041] By the above method, the optimal primer pair screened by the present invention is as follows:

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

[0043] Reverse primer: CEBPAfull-R (the sequence is 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 follows. Figure 2 In it, lane 1 is DL1000bp DNA marker; lane 14 is DL5000bp DNA marker; lanes 2 - 13 are 12 randomly selected samples, and lane 12 is a blank control (non-amplified sample). The DL1000bp DNA marker consists of DNA fragments 1000bp, 700bp, 500bp, 400bp, 300bp, 200bp, 100bp, a total of 7 bands; the DL5000bp DNA marker consists of DNA fragments 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp, a total of 9 bands. From the Figure 2 bands shown in lanes 2 - 11 and 13, it can be seen that the sizes of the PCR products are consistent, indicating that the optimal primer pair provided by the present invention can amplify the full length of the CEBPA gene coding region and there are no non-specific bands.

[0045] Example 2: Probe

[0046] The present invention provides a probe for detecting whether there is a biallelic gene mutation in the CEBPA point mutation. By introducing a single mutation on both sides of the N-terminal where a single base mutation occurs in the CEBPA gene, the disadvantage that T7EI has poor recognition ability for single base mutations is avoided. The design idea is as follows:

[0047] Retrieve the common mutation types in the coding region of CEBPA in the COSMIC database (https: / / cancer.sanger.ac.uk / ). It is found that single-base changes are mainly observed at the N-terminus of the gene, while two- or three-base changes are mainly seen at the C-terminus. According to the characteristics of the enzymes used in the experiment, single mutations are introduced on both sides of the gene where single-base mutations often occur at the N-terminus (for example, if the original sequence is AACTCCG and the mutated sequence is AACGGCCG, the sequence after artificial introduction of the mutation is AATTACG). The mutated sequence is artificially synthesized and named CEBPA-mut (the sequence is shown as SEQ ID No: 13), which is used as an auxiliary detection tool. Submit the mutated sequence to be synthesized to a biological company for synthesis. The biological company finally provides a cloned strain and plasmid dry powder containing the target fragment. The artificial synthesis of the gene sequence can be consulted with a professional biological company for operation.

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

[0049] Since no sufficient samples with double mutations occurring on the same DNA strand are found, in order to detect the feasibility of the technical method, the test samples need to be prepared according to the following method.

[0050] First step: Combine the existing specimens in the laboratory, select the samples with the single mutation of c.937-939dup found by NGS or Sanger sequencing, extract the sample DNA, and adjust the DNA concentration to 30-50 ng / μL to obtain the sample DNA template.

[0051] Second step: Dissolve the upstream and downstream primers shown in Table 1 below into a 10 μM working solution, and mix them in equal volumes to prepare a primer mixture. The labels of the primer mixture correspond one by one to the reaction tube labels.

[0052] Third step: Take eight transparent 200 μL reaction tubes, label them as 1, 2, 3, 4, 5, 6, 7, and 8 in sequence. Add 10 μL of 2× reaction solution, 4 μL of 5× PCR enhancer, 2 μL of ultrapure water, 2 μL of the primer mixture, and 2 μL of the sample DNA template to them, and centrifuge briefly after mixing. Among them, the primer mixture added to each tube corresponds one by one to the reaction tube number.

[0053] Table 1. Amplification primer table

[0054]

[0055]

[0056] Remarks:

[0057] The sequence of c.58CT-F is shown as 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 amplifier, and 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; (3) Annealing and extension at 72°C for 90 s; (4) Repeat steps (2)-(3) for 35 cycles; (5) Extension at 72°C for 7 min; (6) Store at 4°C.

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

[0069] Step 6: Take 4 transparent 200 μL reaction tubes, label them as 9, 10, 11, and 12 in sequence, 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) to them and mix.

[0070] Step 7: Mix the amplified Reaction Solutions 1 and 2 in equal volumes to obtain Mixture 1, mix Reaction Solutions 3 and 4 in equal volumes to obtain Mixture 2, mix Reaction Solutions 5 and 6 in equal volumes to obtain Mixture 3, and mix Reaction Solutions 7 and 8 in equal volumes to obtain Mixture 4; Add 2 μL of Mixture 1 to Reaction Tube 9, add 2 μL of Mixture 2 to Reaction Tube 10, add 2 μL of Mixture 3 to Reaction Tube 11, and add 2 μL of Mixture 4 to Reaction Tube 12 to make the final volume of the reaction solution in each tube 20 μL. After mixing, centrifuge briefly.

[0071] Step 8: Place Reaction Tubes 9 - 12 in a PCR amplifier. The PCR amplification reaction conditions are as follows: (1) Pre-denature at 95°C for 5 min; (2) Denature at 95°C for 30 s; (3) Anneal and extend at 72°C for 90 s; (4) Repeat steps (2) - (3) for 35 cycles; (5) Extend at 72°C for 7 min; (6) Store at 4°C.

[0072] Step 9: Prepare 2% agarose and perform DNA electrophoresis analysis on the amplification products in Reaction Tubes 9 - 12. The results are as Figure 4 shown. Figure 4 In Lane 5 is the DL1000 DNA marker; Lanes 1 - 4 are the amplification fragments of Reaction Tubes 9, 10, 11, and 12. The size of the PCR products is consistent with the expectation, and there are no non-specific bands.

[0073] The reaction solution in Reaction Tube 9 is the PCR product of the sample where the c.58CT and c.937 - 939dup mutations are on the same chromosome.

[0074] The reaction solution in Reaction Tube 10 is the PCR product of the sample where the c.68del and c.937 - 939dup mutations are on the same chromosome.

[0075] The reaction solution in Reaction Tube 11 is the PCR product of the sample where the c.179dup and c.937 - 939dup mutations are on the same chromosome.

[0076] The reaction solution in Reaction Tube 12 is the PCR product of the sample where the c.247del and c.937 - 939dup mutations are on the same chromosome.

[0077] Example 4: Detection of CEBPA Biallelic Gene Mutations

[0078] See Figure 1, the principle of the detection method for CEBPA biallelic gene mutations provided by the present invention is as follows: First, the corresponding PCR products are amplified from the test samples respectively. Through high-temperature denaturation by heat treatment and slow cooling for annealing, they are randomly paired into DNA double strands. Then, taking advantage of the characteristics of T7E1 enzyme, it recognizes and cuts the incompletely paired DNA. According to the different positions of the mismatches, the PCR products are cut into different fragment lengths. Finally, based on the sizes of the bands in agarose gel electrophoresis, it is determined whether the two mutations are on the same DNA strand. (Note: Figure 1 In the figure, lane M represents the marker, and lane CT represents the blank control).

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

[0080] The first step: Prepare the annealing reaction system. Take a transparent 200 μL reaction tube, 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 test sample PCR product to make the volume of the liquid in the reaction tube 19 μL. After mixing, centrifuge briefly.

[0081] The second step: Conduct the annealing reaction. Place the reaction tube into a PCR amplifier, and set the reaction conditions as follows: (1) Pre-denature at 95°C for 5 min; (2) Annealing one: Cool from 95°C to 85°C at a rate of 0.2°C / s; (3) Annealing two: Cool from 85°C to 25°C at a rate of 0.1°C / s; (4) Store at 4°C.

[0082] The third step: Take out the system after the annealing reaction in the second step from the PCR instrument, add 1 μL of T7EI to the reaction tube, and then place the reaction tube into the PCR amplifier again. Set the reaction conditions as follows: (1) Incubate with enzyme at 37°C for 40 min; (2) Terminate the enzyme digestion reaction at 85°C for 15 min; (3) Store at 4°C.

[0083] The fourth step: Prepare 2% agarose, perform agarose gel electrophoresis on the enzyme digestion products in the reaction tube, take pictures and observe, and analyze the enzyme digestion effect.

[0084] Among them, the 10×T7EI reaction buffer is a commercially available product; the CEBPA-mut PCR product is obtained by extracting the probe plasmid solution in Example 2 and using this 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 test sample PCR product is obtained by performing 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 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 it in a shaker for 14 h (37 °C, 220 rpm);

[0087] (2) Use a commercial plasmid extraction kit to extract the plasmid of CEBPA-mut from the above culture solution and dilute its concentration to 10 6 / μL.

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

[0089] According to the database and laboratory test data, select double mutant samples of two mutation types for testing (c.58C>T and c.937-939dup; c.68del and c.937-939dup). Among them, it is known that the reaction solution in tube 9 in Example 3 is the PCR product of the sample with c.58C>T and c.937-939dup mutations on the same chromosome; the reaction solution in tube 10 is the PCR product of the sample with c.68del and c.937-939dup mutations on the same chromosome.

[0090] The test results are shown in Figure 5 As shown, lanes 1, 9, and 17 are DL1000 DNA markers, lanes 8 and 16 are the full-length gene fragments without being digested by enzymes, lanes 2, 4, 6, 10, 11, and 14 are the electrophoresis diagrams of c.58C>T and c.937-939dup mutations on different chromosomes; lanes 3, 5, 7, 12, 13, and 15 are the electrophoresis diagrams of c.58C>T and c.937-939dup mutations on the same chromosome; lanes 18, 26, and 34 are DL1000 DNA markers, lanes 25 and 33 are the full-length gene fragments without being digested by enzymes, lanes 19, 21, 23, 27, 29, and 31 are the electrophoresis diagrams of c.68del and c.937-939dup mutations on different chromosomes; lanes 20, 22, 24, 28, 30, and 32 are the electrophoresis diagrams of c.68del and c.937-939dup mutations on the same chromosome.

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

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

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

[0094] The above are only the preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as a limitation of the present invention. The protection scope of the present invention should be subject to the scope defined by the claims. For those of ordinary skill in the art, without departing from the spirit and scope of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as within the protection scope of the present invention.

Claims

1. A primer and probe for detecting biallelic CEBPA gene mutations, characterized in that: The primers are used for full-length amplification of the CEBPA gene coding region, and include an upstream primer and a downstream primer, wherein 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 primer and probe according to claim 1, characterized in that: The probe has a nucleotide sequence as shown in SEQ ID No:

13.

3. A kit for detecting biallelic CEBPA gene mutations, characterized in that: The kit comprises the primers according to claim 1, the probes according to claim 1 or 2 and T7 endonuclease I.

4. A method for detecting biallelic CEBPA gene mutations, characterized in that: The following steps are involved: Step 1, extracting DNA of the sample to be tested, and using the DNA of the sample to be tested as a template, performing a PCR amplification reaction with the primers described in claim 1 to obtain a PCR product of the sample to be tested; Step 2, extracting the plasmid solution of the probe according to claim 1 or 2, and using the probe plasmid solution as a template and the primers according to claim 1 to perform PCR amplification reaction to obtain a probe PCR product; Step 3: Mix equal volumes of the PCR product of the sample to be tested in step 1 and the probe PCR product in step 2 and perform denaturation annealing. After the denaturation annealing is completed, add T7 endonuclease I for enzyme digestion reaction, and perform agarose gel electrophoresis on the enzyme digestion products. According to the electrophoresis results, determine whether the two mutations of the sample to be tested are biallelic mutations.

5. The method according to claim 4, characterized in that: The method for determining whether the two mutations of 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 the band length is less than 1000bp, the two mutations of the sample to be tested are located on the same chromosome; if the band length is ≥1000bp, the two mutations of the sample to be tested are located on different chromosomes.

6. The method according to claim 4, characterized in that: The procedure of the PCR amplification reaction in step 1 and step 2 comprises the following steps: Pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing and extension at 72°C for 90 s, 35 cycles; extension at 72°C for 7 min; storage at 4°C.

7. The method according to claim 4, characterized in that: The denaturation annealing procedure of 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.

8. The method according to claim 4, characterized in that: The enzyme digestion reaction in step 3 comprises the following steps: incubating the enzyme digestion at 37° C. for 40 min; terminating the enzyme digestion reaction at 85° C. for 15 min; and storing at 4° C.

9. The method according to claim 4, characterized in that: In the 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.

10. The method according to claim 4, characterized in that: In step 2, 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 of each of the upstream and downstream primers with a concentration of 10 μM, and 1 μL of each of the upstream and downstream primers with a concentration of 10 μM. 6 / μL of probe plasmid liquid template 2μL.

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