A primer probe and kit for detecting H3F3A gene mutation and its application

Abstract

The invention discloses a primer probe and a kit for detecting H3F3A gene mutation and its application, wherein the H3F3A gene mutation type is K27M, G34R and G34V mutation. The kit comprises a set of primer probe groups for detecting H3F3A gene mutation and a universal reference gene primer probe group, wherein the primer probe group for detecting H3F3A gene mutation comprises specific upstream primers for K27M, G34R and G34V mutations, a universal downstream primer and a universal fluorescent probe respectively. The kit provided by the invention can detect common mutation types H3F3A K27M, H3F3A G34R and H3F3A G34V in the H3F3A gene, has the advantages of high sensitivity, convenient and fast operation and low cost, and has broad clinical application prospects in diagnosing diseases such as diffuse midline glioma and diffuse cerebral hemispheric glioma.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular detection of gene mutations, and in particular to a primer probe and a kit for detecting H3F3A gene mutations and applications thereof. Background Art

[0002] The 2021 edition of the WHO Classification of Tumors of the Central Nervous System divides gliomas into grades 1 to 4, with grades 1 and 2 being low-grade gliomas and grades 3 and 4 being high-grade gliomas. Diagnosis of gliomas requires obtaining specimens through tumor resection or biopsy surgery, performing an integrated diagnosis of histopathology and molecular pathology, and determining pathological grade and molecular subtypes. The median survival of patients with WHO grade II tumors is more than 5 years, 2-3 years for grade III, and 14.6 months for grade IV. Molecular markers, such as H3F3A, TERT gene, IDH1 gene, etc., are of great significance for the classification, typing, grading, prognosis and treatment of gliomas.

[0003] The H3F3A gene (also known as H3.3 or H3-3A) belongs to the histone gene family. Common mutation types of the H3F3A gene are H3F3A p.K28M (K27M) and p.G35R / V (G34R / G34V), which are key reference indicators for diagnosing diffuse midline glioma and diffuse cerebral hemispheric glioma, respectively.

[0004] H3F3A gene mutation detection is of great significance for the clinical management of central nervous system tumors and has been included in a number of clinical diagnosis and treatment guidelines, such as the "2021 WHO Central Nervous System Tumor Classification Overview", the "2016 NCCN Oncology Clinical Practice Guidelines: Central Nervous System Tumors" and the "Guidelines for the Diagnosis and Treatment of Glioma (2022 Edition)".

[0005] At present, the main method for detecting H3F3A gene mutation is qPCR. The prior art CN105734137A uses a primer probe modified with locked nucleic acid LNA for detection, but the detection cost of the LNA modification method is high and the effect of detecting mutations with low mutation frequency is not good. Summary of the invention

[0006] The technical problem to be solved by the present invention is the above-mentioned defects and shortcomings in the prior art, and a primer probe and a kit for detecting H3F3A gene mutation and their application are provided.

[0007] The first object of the present invention is to provide a universal downstream primer for detecting H3F3A gene mutation.

[0008] The second object of the present invention is to provide a universal fluorescent probe for detecting H3F3A gene mutations.

[0009] The third object of the present invention is to provide a composition for detecting H3F3A gene mutation.

[0010] The fourth object of the present invention is to provide the use of the universal downstream primer, universal fluorescent probe and / or combination mentioned above in the preparation of a product for detecting H3F3A gene mutation.

[0011] The fifth object of the present invention is to provide a kit for detecting H3F3A gene mutation.

[0012] The sixth object of the present invention is to provide a reaction system for detecting H3F3A gene mutation.

[0013] In order to achieve the above object, the present invention is implemented by the following scheme:

[0014] A universal downstream primer for detecting H3F3A gene mutation, the nucleotide sequence of the universal downstream primer is shown in SEQ ID NO: 2, and the H3F3A gene mutation is H3F3A K27M, H3F3AG34R and H3F3A G34V mutation.

[0015] A universal fluorescent probe for detecting H3F3A gene mutations, the nucleotide sequence of the universal fluorescent probe is shown in SEQ ID NO: 3, and the H3F3A gene mutations are H3F3A K27M, H3F3AG34R and H3F3A G34V mutations.

[0016] Preferably, the 5' end of the universal fluorescent probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group.

[0017] As an practicable manner, the 5' end of the universal fluorescent probe is labeled with FAM, and the 3' end is labeled with MGB.

[0018] A composition for detecting H3F3A gene mutations, the composition comprising the universal downstream primer and the universal fluorescent probe, the H3F3A gene mutations being H3F3A K27M, H3F3A G34R and H3F3A G34V mutations.

[0019] Preferably, the composition further comprises one or more of the upstream primers of the nucleotide sequence such as SEQ ID NO: 1, the nucleotide sequence such as SEQ ID NO: 7 and the nucleotide sequence such as SEQ ID NO: 8.

[0020] More preferably, the upstream primer of the nucleotide sequence such as SEQ ID NO: 1 is used to detect the H3F3A K27M gene mutation, the upstream primer of the nucleotide sequence such as SEQ ID NO: 7 is used to detect the H3F3A G34R gene mutation, and the upstream primer of the nucleotide sequence such as SEQ ID NO: 8 is used to detect the H3F3A G34V gene mutation.

[0021] More preferably, the composition further comprises internal reference gene primers having nucleotide sequences as shown in SEQ ID NO:4 and SEQ ID NO:5 and an internal reference gene probe having a nucleotide sequence as shown in SEQ ID NO:6.

[0022] More preferably, the 5' end of the internal reference gene probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group.

[0023] As an practicable manner, the 5' end of the internal reference gene probe is labeled with VIC, and the 3' end is labeled with MGB.

[0024] The use of the universal downstream primer, universal fluorescent probe and / or combination in the preparation of a product for detecting H3F3A gene mutations, wherein the H3F3A gene mutations are H3F3A K27M, H3F3A G34R and H3F3AG34V mutations.

[0025] A kit for detecting H3F3A gene mutations, the kit comprising the universal downstream primers and / or universal fluorescent probes, the H3F3A gene mutations being H3F3A K27M, H3F3A G34R and H3F3A G34V mutations.

[0026] Preferably, the kit further comprises one or more of upstream primers of a nucleotide sequence such as SEQ ID NO: 1, a nucleotide sequence such as SEQ ID NO: 7, and a nucleotide sequence such as SEQ ID NO: 8.

[0027] Preferably, the kit further comprises internal reference gene primers having nucleotide sequences as shown in SEQ ID NO:4 and SEQ ID NO:5, an internal reference gene probe having a nucleotide sequence as shown in SEQ ID NO:6, 10× reaction buffer, dNTPs, TaqDNA polymerase, a positive control reference and / or a negative control reference.

[0028] More preferably, the positive control reference substance is a standard substance with H3F3A K27M, H3F3A G34R and H3F3AG34V gene mutation frequencies of 5%, 2%, 1%, 0.5% and 0%, respectively.

[0029] More preferably, the negative control reference substance is sterile ultrapure water.

[0030] A reaction system for detecting H3F3A gene mutations, wherein the concentrations of an upstream primer and a universal downstream primer are respectively 0.1-0.4 μM, the concentration of a universal fluorescent probe is 0.1-0.4 μM, the concentration of an internal reference gene primer is 0.05-0.2 μM, the concentration of an internal reference gene probe is 0.02-0.08 μM, 10× reaction buffer is 4.8-5.2 μl, dNTPs is 0.1-0.3 mM, Taq DNA polymerase is 2-3 U, a sample to be tested is 4-6 ng / μl, and deionized water without RNase is added to 50 μl.

[0031] More preferably, the reaction system is: the concentrations of the upstream primer and the universal downstream primer are 0.2 μM respectively, the concentration of the universal fluorescent probe is 0.2 μM, the concentration of the internal reference gene primer is 0.1 μM, the concentration of the internal reference gene probe is 0.05 μM, 10× reaction buffer 5 μl, dNTPs 0.2 mM, Taq DNA polymerase 2.5 U, the sample to be tested 5 ng / μl, and deionized water without RNase is added to 50 μl.

[0032] Preferably, the reaction conditions of the reaction system are 95°C for 30s; 95°C for 20s, 65°C for 30s, 10 cycles; 95°C for 20s, 60°C for 30s, 35 cycles.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention provides a primer probe and a kit for detecting H3F3A gene mutation, which can detect common mutation types H3F3A K27M, H3F3A G34R and H3F3A G34V in the H3F3A gene, and has the advantages of high sensitivity, convenient and fast operation, and low cost.

[0035] (1) High sensitivity: For samples containing the H3F3A K27M gene mutation, samples with a K27M gene mutation frequency of 0.5% can be detected; for samples containing the H3F3A G34R gene mutation, samples with a G34R gene mutation frequency of 0.5% can be detected; for samples containing the H3F3A G34V gene mutation, samples with a G34V gene mutation frequency of 0.5% can be detected.

[0036] (2) Convenient operation: The operation steps are simple and can directly detect H3F3A gene mutations. Negative and positive results can be directly judged by observing the amplification curve. The results are direct and objective. No complicated electrophoresis or other analysis steps are required, and the operator's experience is not high.

[0037] (3) Lower cost: Compared with CN105734137A which uses a primer probe modified with locked nucleic acid (LNA) with higher detection cost, the present invention provides an economical and simple primer probe and kit for H3F3A gene mutation detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the detection result of H3F3A K27M primer probe set 1.

[0039] Figure 2 This is the detection result of H3F3A K27M primer probe set 2.

[0040] Figure 3 This is the detection result of H3F3A G34R primer probe set 1.

[0041] Figure 4 This is the detection result of H3F3A G34R primer probe set 2.

[0042] Figure 5 This is the detection result of H3F3A G34V primer probe set 1.

[0043] Figure 6 This is the detection result of H3F3A G34V primer probe set 2. DETAILED DESCRIPTION

[0044] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0045] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0046] Example 1 Effect of primer probe set on fluorescence quantitative PCR detection of H3F3A K27M gene mutation

[0047] 1. Design primers and probes

[0048] H3F3A (NG_065173.1) was used as the specific target gene and GAPDH (NG_007073.2) was used as the internal reference gene. The primers were designed using the primer design software Primer express and synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0049] K27M Primer Probe Set 1:

[0050] K27M upstream primer: 5'-TACAAAAGCCGCTCGAAT-3' (SEQ ID NO: 9);

[0051] Universal downstream primer: 5'-TTTGTTAAATTATGTGGATACAT-3' (SEQ ID NO: 10);

[0052] Universal fluorescent probe: 5'-CAAGAGAGACTTTGTCC-3' (SEQ ID NO: 11);

[0053] The universal fluorescent probe is labeled with the fluorescent group FAM at the 5' end and the quenching group MGB at the 3' end.

[0054] K27M Primer Probe Set 2:

[0055] K27M upstream primer: 5'-TACAAAAGCCGCTCGTAT-3' (SEQ ID NO: 1);

[0056] Universal downstream primer: 5′-GATACATACAAGAGAGACTTTGTC-3′ (SEQ ID NO: 2);

[0057] Universal fluorescent probe: 5'-AAGAAACCTCATCGTTAC-3' (SEQ ID NO: 3);

[0058] The universal fluorescent probe is labeled with the fluorescent group FAM at the 5' end and the quenching group MGB at the 3' end.

[0059] Internal reference gene primer probe set:

[0060] Internal reference gene upstream primer: 5'-TTGATGATGCTGAGTGTACA-3' (SEQ ID NO: 4);

[0061] Downstream primer of internal reference gene: 5'-TTGTACATGGTATTCACCACC-3' (SEQ ID NO: 5);

[0062] Internal reference gene probe: 5'-CAAGCGTTTTCTCCCT-3' (SEQ ID NO: 6);

[0063] The 5' end of the internal reference gene probe is labeled with the fluorescent group VIC, and the 3' end is labeled with the quenching group MGB.

[0064] 2. Perform PCR reaction

[0065] The PCR reagent was Taq HSDNA polymerase (Cat. No. P132) from Nanjing Novozymes Biotech Co., Ltd. The samples were standard products with H3F3A K27M gene mutation frequencies of 5%, 2%, 1%, 0.5%, and 0%, respectively.

[0066] The reaction system is shown in Table 1:

[0067] Table 1 H3F3A K27M reaction system

[0068]

[0069] The reaction program of primer probe set 1 was: 95°C for 30 s; 95°C for 20 s, 60°C for 30 s, for 45 cycles.

[0070] The reaction program of primer probe set 2 was: 95°C for 30 s; 95°C for 20 s, 65°C for 30 s, 10 cycles; 95°C for 20 s, 60°C for 30 s, 35 cycles.

[0071] 3. Experimental Results

[0072] The results are shown in Table 2.

[0073] Table 2H3F3A K27M test results

[0074]

[0075] Figure 1 This is the result of the H3F3A K27M primer probe set 1 detection. Figure 2 This is the result of the H3F3A K27M primer probe set 2 detection. Figure 1 and Figure 2 It can be seen that the detection effects of different primer probe sets are significantly different, and primer probe set 2 has a higher sensitivity.

[0076] Example 2 Effect of primer probe set on fluorescence quantitative PCR detection of H3F3A G34R gene mutation

[0077] 1. Design primers and probes

[0078] H3F3A (NG_065173.1) was used as the specific target gene and GAPDH (NG_007073.2) was used as the internal reference gene. The primers were designed using the primer design software Primer express and synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0079] G34R Primer Probe Set 1:

[0080] G34R upstream primer: 5'-GTGCGCCCTCTACTGAAA-3' (SEQ ID NO: 12);

[0081] Universal downstream primer: 5'-TTTGTTAAATTATGTGGATACAT-3' (SEQ ID NO: 13);

[0082] Universal fluorescent probe: 5'-CAAGAGAGACTTTGTCC-3' (SEQ ID NO: 14);

[0083] The universal fluorescent probe is labeled with the fluorescent group FAM at the 5' end and the quenching group MGB at the 3' end.

[0084] G34R Primer Probe Set 2:

[0085] G34R upstream primer: 5'-GTGCGCCCTCTACTGCAA-3' (SEQ ID NO: 7);

[0086] Universal downstream primer: 5′-GATACATACAAGAGAGACTTTGTC-3′ (SEQ ID NO: 2);

[0087] Universal fluorescent probe: 5'-AAGAAACCTCATCGTTAC-3' (SEQ ID NO: 3);

[0088] The universal fluorescent probe is labeled with the fluorescent group FAM at the 5' end and the quenching group MGB at the 3' end.

[0089] Internal reference gene primer probe set:

[0090] Internal reference gene upstream primer: 5'-TTGATGATGCTGAGTGTACA-3' (SEQ ID NO: 4);

[0091] Downstream primer of internal reference gene: 5'-TTGTACATGGTATTCACCACC-3' (SEQ ID NO: 5);

[0092] Internal reference gene probe: 5'-CAAGCGTTTTCTCCCT-3' (SEQ ID NO: 6);

[0093] The 5' end of the internal reference gene probe is labeled with the fluorescent group VIC, and the 3' end is labeled with the quenching group MGB.

[0094] 2. Perform PCR reaction

[0095] The PCR reagent was Taq HSDNA polymerase (Cat. No. P132) from Nanjing Novozymes Biotech Co., Ltd. The samples were standard products with H3F3A G34R gene mutation frequencies of 5%, 2%, 1%, 0.5%, and 0%, respectively.

[0096] The reaction system is shown in Table 3:

[0097] Table 3H3F3A G34R reaction system

[0098]

[0099] The reaction program of primer probe set 1 was: 95°C for 30 s; 95°C for 20 s, 60°C for 30 s, for 45 cycles.

[0100] The reaction program of primer probe set 2 was: 95°C for 30 s; 95°C for 20 s, 65°C for 30 s, 10 cycles; 95°C for 20 s, 60°C for 30 s, 35 cycles.

[0101] 3. Experimental Results

[0102] The results are shown in Table 4.

[0103] Table 4 H3F3A G34R test results

[0104]

[0105] Figure 3 This is the result of the H3F3A G34R primer probe set 1 detection. Figure 4 This is the result of the H3F3A G34R primer probe set 2 detection. Figure 3 and Figure 4 It can be seen that the detection effects of different primer probe sets are significantly different, and primer probe set 2 has a higher sensitivity.

[0106] Example 3 Effect of primer probe set on fluorescence quantitative PCR detection of H3F3A G34V gene mutation

[0107] 1. Design primers and probes

[0108] H3F3A (NG_065173.1) was used as the specific target gene and GAPDH (NG_007073.2) was used as the internal reference gene. The primers were designed using the primer design software Primer express and synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0109] G34V Primer Probe Set 1:

[0110] G34V upstream primer: 5′-TGCGCCCTCTACTGGGGT-3′ (SEQ ID NO: 15);

[0111] Universal downstream primer: 5'-TTTGTTAAATTATGTGGATACAT-3' (SEQ ID NO: 16);

[0112] Universal fluorescent probe: 5'-CAAGAGAGACTTTGTCC-3' (SEQ ID NO: 17);

[0113] The universal fluorescent probe is labeled with the fluorescent group FAM at the 5' end and the quenching group MGB at the 3' end.

[0114] G34V Primer Probe Set 2:

[0115] G34V upstream primer: 5'-TGCGCCCTCTACTGAAGT-3' (SEQ ID NO: 8);

[0116] Universal downstream primer: 5′-GATACATACAAGAGAGACTTTGTC-3′ (SEQ ID NO: 2);

[0117] Universal fluorescent probe: 5'-AAGAAACCTCATCGTTAC-3' (SEQ ID NO: 3);

[0118] The universal fluorescent probe is labeled with the fluorescent group FAM at the 5' end and the quenching group MGB at the 3' end.

[0119] Internal reference gene primer probe set:

[0120] Internal reference gene upstream primer: 5'-TTGATGATGCTGAGTGTACA-3' (SEQ ID NO: 4);

[0121] Downstream primer of internal reference gene: 5'-TTGTACATGGTATTCACCACC-3' (SEQ ID NO: 5);

[0122] Internal reference gene probe: 5'-CAAGCGTTTTCTCCCT-3' (SEQ ID NO: 6);

[0123] The 5' end of the internal reference gene probe is labeled with the fluorescent group VIC, and the 3' end is labeled with the quenching group MGB.

[0124] 2. Perform PCR reaction

[0125] The PCR reagent was Taq HSDNA polymerase (Cat. No. P132) from Nanjing Novozymes Biotech Co., Ltd. The samples were standard products with H3F3A G34V gene mutation frequencies of 5%, 2%, 1%, 0.5%, and 0%, respectively.

[0126] The reaction system is shown in Table 5:

[0127] Table 5 H3F3A G34V reaction system

[0128]

[0129] The reaction program of primer probe set 1 was: 95°C for 30 s; 95°C for 20 s, 60°C for 30 s, for 45 cycles.

[0130] The reaction program of primer probe set 2 was: 95°C for 30 s; 95°C for 20 s, 65°C for 30 s, 10 cycles; 95°C for 20 s, 60°C for 30 s, 35 cycles.

[0131] 3. Experimental Results

[0132] The results are shown in Table 6.

[0133] Table 6 H3F3A G34V test results

[0134]

[0135]

[0136] Figure 5 This is the result of the H3F3A G34V primer probe set 1 test. Figure 6 This is the result of the H3F3A G34V primer probe set 2 detection. Figure 5 and Figure 6 It can be seen that the detection effects of different primer probe sets are significantly different, and primer probe set 2 has a higher sensitivity.

[0137] Example 4 A fluorescent quantitative PCR detection method for detecting H3F3A gene mutation

[0138] 1. Preparation of DNA Samples

[0139] Extraction of genomic DNA: Formalin-fixed paraffin-embedded tissue samples (FFPE) were used as test specimens, and FFPE nucleic acid extraction reagent was used to extract genomic DNA.

[0140] Dilute genomic DNA: Dilute genomic DNA with nuclease-free water.

[0141] 2. Perform Fluorescence Quantitative PCR Reaction

[0142] Prepare a fluorescent quantitative PCR reaction system, as shown in Table 7. The upstream primer is one of K27M upstream primer (SEQ ID NO: 1), G34R upstream primer (SEQ ID NO: 7) or G34V upstream primer (SEQ ID NO: 8).

[0143] The K27M upstream primer (SEQ ID NO: 1) and the universal downstream primer (SEQ ID NO: 2) were used to detect the H3F3AK27M gene mutation, the G34R upstream primer (SEQ ID NO: 7) and the universal downstream primer (SEQ ID NO: 2) were used to detect the H3F3A G34R gene mutation, and the G34V upstream primer (SEQ ID NO: 8) and the universal downstream primer (SEQ ID NO: 2) were used to detect the H3F3A G34V gene mutation.

[0144] Table 7 Fluorescence quantitative PCR reaction system

[0145]

[0146]

[0147] ABI 7500 was used for PCR amplification, and the detection channels were set to FAM and VIC. The PCR reaction program was set to 95°C for 30 s; 95°C for 20 s, 65°C for 30 s, 10 cycles; 95°C for 20 s, 60°C for 30 s, 35 cycles. ABI 7500 PassiveReference was set to None.

[0148] 3. Interpretation of results

[0149] 1. Determination of test effectiveness:

[0150] 1) Positive control: The FAM and VIC channels of each reaction tube have obvious amplification curves, and the Ct value is ≤25.

[0151] 2) Negative control: There should be no amplification curve in the FAM and VIC channels.

[0152] The above requirements must be met simultaneously in the same test, otherwise the test will be invalid and must be repeated.

[0153] 2. Determination of the validity of test samples:

[0154] Internal reference gene: In all samples tested, the VIC channel had an obvious amplification curve, and the Ct value was ≤25.

[0155] 1) If the Ct value is less than 15, it means that the sample genomic DNA is excessively added. If there is no obvious amplification curve in the FAM and VIC channels of all reaction tubes or the result is negative according to the interpretation criteria, there is no need to retest. Otherwise, it is recommended to dilute and retest;

[0156] 2) If 15≤Ct value≤25, it means that the amount of sample genomic DNA added is appropriate;

[0157] 3) If the Ct value is greater than 25, it means that the genomic DNA content of the sample is too low, and only samples with a high mutation frequency can detect mutations. It is necessary to re-prepare the sample or increase the sample amount.

[0158] 3. Determination of test results:

[0159] After determining the validity of the test sample, the sample test results are judged according to the following standards to determine whether there are mutations at each test site of the sample.

[0160] 1) If there is no obvious amplification curve in the FAM and VIC channels of each detection site, the mutation of the site is negative or below the detection limit of this kit;

[0161] 2) If the FAM and VIC channels of each detection site have obvious amplification curves, calculate the △Ct value of the site. If the △Ct of the site is ≤12, the sample is positive for the mutation corresponding to the site; otherwise, it is negative for the mutation corresponding to the site or below the detection limit of this kit (see Table 8 for details).

[0162] △Ct value = mutation Ct value FAM -Internal reference Ct value VIC , the mutation Ct value refers to the Ct value corresponding to the sample mutation signal (FAM), and the internal reference Ct value refers to the Ct value corresponding to the sample internal reference gene signal (VIC).

[0163] Table 8 Result judgment criteria

[0164] Reaction solution Mutation type ΔCt value Positive Negative H3F3A reaction solution p.K28M(K27M) 12 △Ct≤12 △Ct>12 H3F3A reaction solution p.G35R / V(G34R / G34V) 12 △Ct≤12 △Ct>12

[0165] Example 5 A fluorescent quantitative PCR detection kit for detecting H3F3A gene mutation

[0166] 1. Composition

[0167] 1. A set of fluorescent quantitative PCR primers and probes for detecting H3F3A gene mutations

[0168] K27M upstream primer: 5'-TACAAAAGCCGCTCGTAT-3' (SEQ ID NO: 1);

[0169] G34R upstream primer: 5'-GTGCGCCCTCTACTGCAA-3' (SEQ ID NO: 7);

[0170] G34V upstream primer: 5'-TGCGCCCTCTACTGAAGT-3' (SEQ ID NO: 8);

[0171] Universal downstream primer: 5′-GATACATACAAGAGAGACTTTGTC-3′ (SEQ ID NO: 2);

[0172] Universal fluorescent probe: 5'-AAGAAACCTCATCGTTAC-3' (SEQ ID NO: 3);

[0173] The universal fluorescent probe is labeled with the fluorescent group FAM at the 5' end and the quenching group MGB at the 3' end.

[0174] 2. A set of reference gene primers and probes for detecting H3F3A gene mutations

[0175] Internal reference gene upstream primer: 5'-TTGATGATGCTGAGTGTACA-3' (SEQ ID NO: 4);

[0176] Downstream primer of internal reference gene: 5'-TTGTACATGGTATTCACCACC-3' (SEQ ID NO: 5);

[0177] Internal reference gene probe: 5'-CAAGCGTTTTCTCCCT-3' (SEQ ID NO: 6);

[0178] The 5' end of the internal reference gene probe is labeled with the fluorescent group VIC, and the 3' end is labeled with the quenching group MGB.

[0179] 3. The PCR reagent was Taq HSDNA polymerase (Cat. No. P132) from Nanjing Novozymes Biotech Co., Ltd.

[0180] The positive control reference substances were standards with H3F3A K27M, H3F3A G34R and H3F3A G34V gene mutation frequencies of 5%, 2%, 1%, 0.5% and 0%.

[0181] The negative control reference was sterile ultrapure water.

[0182] 2. Usage

[0183] The detection and result interpretation were performed according to the method of Example 4.

[0184] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A composition for detecting H3F3A K27M gene mutation, characterized in that: The composition comprises a universal downstream primer, a universal fluorescent probe and an upstream primer; The nucleotide sequence of the universal downstream primer is shown in SEQ ID NO: 2; The nucleotide sequence of the universal fluorescent probe is shown in SEQ ID NO: 3; The nucleotide sequence of the upstream primer is shown in SEQ ID NO:

1.

2. A composition for detecting H3F3A G34R gene mutation, characterized in that: The composition comprises a universal downstream primer, a universal fluorescent probe and an upstream primer; The nucleotide sequence of the universal downstream primer is shown in SEQ ID NO: 2; The nucleotide sequence of the universal fluorescent probe is shown in SEQ ID NO: 3; The nucleotide sequence of the upstream primer is shown in SEQ ID NO:

7.

3. A composition for detecting H3F3A G34V gene mutation, characterized in that: The composition comprises a universal downstream primer, a universal fluorescent probe and an upstream primer; The nucleotide sequence of the universal downstream primer is shown in SEQ ID NO: 2; The nucleotide sequence of the universal fluorescent probe is shown in SEQ ID NO: 3; The nucleotide sequence of the upstream primer is shown in SEQ ID NO:

8.

4. Use of the composition according to claim 1 in preparing a product for detecting H3F3A K27M gene mutation.

5. Use of the composition according to claim 2 in preparing a product for detecting H3F3A G34R gene mutation.

6. Use of the composition according to claim 3 in preparing a product for detecting H3F3A G34V gene mutation.

7. A kit for detecting H3F3A K27M gene mutation, characterized in that: The kit comprises the composition of claim 1.

8. A kit for detecting H3F3A G34R gene mutation, characterized in that: The kit comprises the composition of claim 2.

9. A kit for detecting H3F3A G34V gene mutation, characterized in that: The kit comprises the composition of claim 3.

10. The kit according to any one of claims 7 to 9, characterized in that The kit also includes internal reference gene primers with nucleotide sequences as shown in SEQ ID NO:4 and SEQ ID NO:5, an internal reference gene probe with a nucleotide sequence as shown in SEQ ID NO:6, 10× reaction buffer, dNTPs, Taq DNA polymerase, a positive control reference substance and / or a negative control reference substance.

11. A reaction system for detecting H3F3A K27M gene mutation, characterized in that: In the reaction system, the concentrations of the upstream primer and the universal downstream primer are 0.1-0.4 μM, the concentration of the universal fluorescent probe is 0.1-0.4 μM, the concentration of the internal reference gene primer is 0.05-0.2 μM, the concentration of the internal reference gene probe is 0.02-0.08 μM, 10× reaction buffer 4.8-5.2 μl, dNTPs 0.1-0.3 mM, Taq DNA polymerase 2-3 U, the sample to be tested 4-6 ng / μl, and RNase-free deionized water is added to 50 μl; The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 1; The nucleotide sequence of the universal downstream primer is shown in SEQ ID NO: 2; The nucleotide sequence of the universal fluorescent probe is shown in SEQ ID NO:

3.

12. A reaction system for detecting H3F3A G34R gene mutation, characterized in that: In the reaction system, the concentrations of the upstream primer and the universal downstream primer are 0.1-0.4 μM, the concentration of the universal fluorescent probe is 0.1-0.4 μM, the concentration of the internal reference gene primer is 0.05-0.2 μM, the concentration of the internal reference gene probe is 0.02-0.08 μM, 10× reaction buffer 4.8-5.2 μl, dNTPs 0.1-0.3 mM, Taq DNA polymerase 2-3 U, the sample to be tested 4-6 ng / μl, and RNase-free deionized water is added to 50 μl; The nucleotide sequence of the upstream primer is shown in SEQ ID NO:7; The nucleotide sequence of the universal downstream primer is shown in SEQ ID NO: 2; The nucleotide sequence of the universal fluorescent probe is shown in SEQ ID NO:

3.

13. A reaction system for detecting H3F3A G34V gene mutation, characterized in that: In the reaction system, the concentrations of the upstream primer and the universal downstream primer are 0.1-0.4 μM, the concentration of the universal fluorescent probe is 0.1-0.4 μM, the concentration of the internal reference gene primer is 0.05-0.2 μM, the concentration of the internal reference gene probe is 0.02-0.08 μM, 10× reaction buffer 4.8-5.2 μl, dNTPs 0.1-0.3 mM, Taq DNA polymerase 2-3 U, the sample to be tested 4-6 ng / μl, and RNase-free deionized water is added to 50 μl; The nucleotide sequence of the upstream primer is shown in SEQ ID NO:8; The nucleotide sequence of the universal downstream primer is shown in SEQ ID NO: 2; The nucleotide sequence of the universal fluorescent probe is shown in SEQ ID NO:

3.

14. The reaction system according to any one of claims 11 to 13, characterized in that: The reaction conditions of the reaction system are 95°C for 30 s; 95°C for 20 s, 65°C for 30 s, 10 cycles; 95°C for 20 s, 60°C for 30 s, 35 cycles.

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