CN110982884A - Primer group, kit and method for detecting AML related gene mutation - Google Patents

Abstract

The invention discloses a primer group, a kit and a method for detecting AML related gene mutation, and belongs to the technical field of gene detection. The primer group provided by the invention comprises one or more of FLT3 gene primer pair, DNMT3A gene primer pair, IDH1 gene primer pair, IDH2 gene first primer pair, IDH2 gene second primer pair, KIT gene primer pair and NPM1 gene primer pair, and the provided KIT comprises the primer group and one or more of FLT3 gene probe, DNMT3A gene probe, IDH1 gene probe, IDH2 gene first probe, IDH2 gene second probe, KIT gene probe and NPM1 gene probe. The detection method for detecting AML related gene mutation provided by the embodiment of the invention has the characteristics of high efficiency, simplicity, convenience, intuition and high sensitivity, and can be used for carrying out quantitative analysis on AML related gene mutation frequency.

Description

Primer group, kit and method for detecting AML related gene mutation

Technical Field

The invention relates to the technical field of gene detection, in particular to a primer group, a kit and a method for detecting AML related gene mutation.

Background

Acute Myeloid Leukemia (AML) is a genetically heterogeneous disease whose clinical course can be predicted by recurrent cytogenetic abnormalities and/or genetic mutations. Cytogenetic detection of specific karyotypic abnormalities is often considered a marker for guiding treatment and determining prognosis. However, karyotypic abnormalities are not detectable in some patients, and these patients have large variation in response to treatment or in prognostic evaluation. With the rapid development of molecular biology techniques, a number of genetic mutations associated with AML progression or AML prognosis have been identified in succession, which fully demonstrates that genetic mutations are important sources of AML pathogenesis.

In AML, IDH mutations are associated with acute myeloid leukemia undifferentiated (subtype M) and normal karyotype acute myeloid leukemia (CN-AML) in FAB typing, and AML patients with IHD mutations have a lower complete remission rate (CR), higher Risk of Relapse (RR) and shorter Overall Survival (OS). In AML patients with cytogenetically normal, the frequency of IDH2(R172) mutations was lower than that of IDH2(R140), and the prognosis of IDH2(R172) mutant patients was worse than that of IDH2(R140) mutant patients. In addition, the risk of relapse for the combination of NPM1, FLT3 ITD, and DNMT3A mutations was much higher than for the NPM1 and FLT3 ITD mutations without DNMT3a6, and there was a trend for lower Overall Survival (OS) for the combination of NPM1, DNMT3A, and IDH 1/2. Whereas exon 8 and exon 17 mutations of the C-KIT gene are more common in AML with t (8; 21) or inv (16), with about 25.8% of exon 8 mutations, about 65% of exon 17 mutations, and mainly the D816V mutation. The gene mutation indicates that the recurrence rate is high and the prognosis is poor. Therefore, it is of great significance to be able to separate patients for prognostic analysis based on the mutation status of different genes.

The BDA (Block Displacement amplification) technology is a temperature-stable polymerase chain reaction, can selectively amplify variant sequences, including Single Nucleotide Variants (SNVs), within about 20 nucleotide windows, and can enable the amplification efficiency of the variant sequences to be 1000 times higher than that of the wild-type sequences. According to known gene mutation sites, specific primers and probes are designed, multiple mutation modes of the same site can be detected at the same annealing temperature, and the gene mutation frequency can be quantitatively analyzed. The Sanger sequencing method is the international gold standard of all the current gene detection, has the specific performance of reaching 100 percent and is a classic technical platform in the aspect of molecular diagnosis. However, Sanger sequencing has low sensitivity and cannot detect samples with low mutation frequency.

In the current method for detecting AML gene mutation at home and abroad, Fluorescence In Situ Hybridization (FISH) can only carry out qualitative detection and has complex operation; the combination of Polymerase Chain Reaction (PCR) technology and Sanger sequencing technology has the limitation of low sensitivity, and is difficult to detect low-frequency mutation; although the NGS technology can be used for qualitative and quantitative detection and has higher sensitivity, the NGS technology has the limitations of long detection time, higher cost, high requirement on low-frequency mutation data volume and the like. Therefore, there is a need to improve the existing AML genetic mutation detection method to provide reference for clinical diagnosis and clinical prognosis treatment of AML.

Disclosure of Invention

The present invention is directed to a primer set, a kit and a method for detecting AML-related gene mutation, which solve the problems of the background art described above.

In order to achieve the above purpose, the embodiments of the present invention provide the following technical solutions:

a primer set for detecting AML-related gene mutation, which comprises one or more of FLT3 gene primer pair, DNMT3A gene primer pair, IDH1 gene primer pair, IDH2 gene first primer pair, IDH2 gene second primer pair, KIT gene primer pair and NPM1 gene primer pair; the nucleotide sequence of the FLT3 gene primer pair is shown in a sequence table SEQ ID NO 1-2; the nucleotide sequence of the DNMT3A gene primer pair is shown as a sequence table SEQ ID NO. 4-5; the nucleotide sequence of the IDH1 gene primer pair is shown in a sequence table SEQ ID NO. 7-8; the nucleotide sequence of the IDH2 gene first primer pair is shown in a sequence table SEQ ID NO. 10-11; the nucleotide sequence of the second primer pair of the IDH2 gene is shown in a sequence table SEQ ID NO. 13-14; the nucleotide sequence of the KIT gene primer pair is shown as a sequence table SEQ ID NO 16-17; the nucleotide sequence of the NPM1 gene primer pair is shown in a sequence table S EQ ID NO: 19-20.

Another objective of an embodiment of the present invention is to provide a kit for detecting AML-related gene mutation, which comprises a PCR amplification reaction reagent, a positive quality control material, a negative quality control material, and a standard curve equation, and the kit further comprises the above primer set and a primer probe set corresponding to the primer set.

Preferably, the primer probe set comprises the primer set and one or more of FLT3 gene probe, DNMT3A gene probe, IDH1 gene probe, IDH2 gene first probe, IDH2 gene second probe, KIT gene probe and NPM1 gene probe; the nucleotide sequence of the FLT3 gene probe is shown in a sequence table SEQ ID NO. 3; the nucleotide sequence of the DNMT3A gene probe is shown in a sequence table SEQ ID NO. 6; the nucleotide sequence of the IDH1 gene probe is shown in a sequence table SEQ ID NO. 9; the nucleotide sequence of the first probe of the IDH2 gene is shown in a sequence table SEQ ID NO. 12; the nucleotide sequence of the second probe of the IDH2 gene is shown in a sequence table SEQ ID NO. 15; the nucleotide sequence of the KI T gene probe is shown in a sequence table SEQ ID NO. 18; the nucleotide sequence of the NPM1 gene probe is shown in a sequence table SEQ ID NO: 21.

Preferably, the molar concentration of each primer in the primer set is 2-100. mu. mol/L.

Preferably, the molar concentration of each primer in the primer probe set is 2-100. mu. mol/L, and the molar concentration of each probe is 10-1000. mu. mol/L.

Preferably, the positive quality control product comprises one or more of FLT3 gene mutation plasmid, DNMT3A gene mutation plasmid, IDH1 gene mutation plasmid, first IDH2 gene mutation plasmid, second IDH2 gene mutation plasmid, KIT gene mutation plasmid and N PM1 gene mutation plasmid.

Preferably, the nucleotide sequence of the FLT3 gene mutant plasmid is shown in a sequence table SEQ ID NO. 22; the nucleotide sequence of the DNMT3A gene mutant plasmid is shown in a sequence table SEQ ID NO. 23; the nucleotide sequence of the IDH1 gene mutant plasmid is shown in a sequence table SEQ ID NO. 24; the nucleotide sequence of the first mutant plasmid of the IDH2 gene is shown in a sequence table SEQ ID NO. 25; the nucleotide sequence of the second mutant plasmid of the IDH2 gene is shown in a sequence table SEQ ID NO: 26; the nucleotide sequence of the KIT gene mutant plasmid is shown as a sequence table SEQ ID NO. 18; the nucleotide sequence of the NPM1 gene mutant plasmid is shown in a sequence table SEQ ID NO: 21.

Preferably, the standard curve equation is obtained by the difference between the first CT value and the second CT value of the standard substance obtained by diluting the mutant plasmid and the healthy human genome DNA by 50%, 10%, 5%, 1%, 0.1% and 0% according to the mutation frequency, and specifically comprises the following steps:

respectively mixing the primer probe set, the PCR amplification reaction reagent and the 6 mutation frequency standard products together for PCR amplification reaction to obtain first CT values of the 6 mutation frequency standard products;

respectively mixing the primer group, the PCR amplification reaction reagent and the 6 mutation frequency standard products together for PCR amplification reaction to obtain second CT values of the 6 mutation frequency standard products;

and obtaining a standard curve equation according to the difference value of the first CT value and the second CT value of the 6 standard products.

It is another object of the embodiments of the present invention to provide a method for detecting mutations in AML-related genes, which comprises the steps of:

obtaining DNA of a sample to be detected as a template;

mixing the primer probe group, the PCR amplification reaction reagent and the template together for PCR amplification reaction to obtain a first CT value;

mixing the primer group, the PCR amplification reaction reagent and the template together for PCR amplification reaction to obtain a second CT value;

carrying out PCR amplification reaction on the negative quality control product to obtain a negative reference CT difference value;

carrying out PCR amplification reaction on the positive quality control product to obtain a positive reference CT difference value;

and comparing the difference value of the first CT value and the second CT value with the positive reference CT difference value and the negative reference CT difference value, and judging whether the gene corresponding to the primer group has mutation.

Preferably, the method further comprises:

sequencing the PCR product obtained by the amplification reaction (such as Sanger sequencing, next-generation high-throughput sequencing, single-molecule sequencing, nanopore sequencing and pyrosequencing) to obtain a sequencing result; if Sanger sequencing is used, direct sequencing can be performed by using a universal primer M13 RP;

analyzing the sequencing result, and identifying the gene mutation type related to AML;

and substituting the difference value of the first CT value and the second CT value into a standard curve equation corresponding to the mutation type according to the identified mutation type to obtain the mutation frequency of the gene corresponding to the primer group.

Compared with the prior art, the embodiment of the invention has the beneficial effects that:

after the polymerase chain reaction provided by the embodiment of the invention selectively amplifies the variant sequence, the sensitivity of Sanger sequencing can be improved from 10% to 0.1%, so that Sanger can detect 0.1% of gene mutation in AML diseases.

(1) The annealing temperatures of all primers designed by the invention are the same, so that the mutation site detection of FLT3, DNMT3A, IDH1, IDH2, KIT and NPM1 genes can be carried out in the same amplification program, and the detection efficiency is high.

(2) The invention can directly sequence the PCR amplification product during detection by adopting the M13RP universal primer, and carry out mutation analysis by software. Because the number of genes to be detected is large, different primers are needed to be used for distinguishing different genes during Sanger sequencing, the workload can be increased and the error rate can be increased in the sequencing reaction, and by using the primer combination provided by the invention, only the M13RP primer is used during sequencing, so that the time is saved, the error rate can be reduced, and the working efficiency is improved.

(3) The detection method of AML-related gene mutation provided by the invention has high sensitivity, can detect mutation frequency as low as 0.1%, and can enrich mutant templates to meet the problem of low sensitivity of Sanger sequencing.

(4) The detection method of AML-related genetic mutation provided by the invention is efficient, simple, convenient, intuitive and high in sensitivity, and can be used for carrying out quantitative analysis on AML-related genetic mutation frequency.

Drawings

FIG. 1 is a standard graph of FLT3 p.D835Y mutation sites obtained in step (7).

FIG. 2 is a standard graph of the DNMT3A p.R882H mutation site obtained in step (7).

FIG. 3 is a standard graph of IDH1 p.R132H mutation sites obtained in step (7).

FIG. 4 is a standard graph of IDH2 p.R140Q mutant sites obtained in step (7).

FIG. 5 is a standard graph of IDH2 p.R172K mutation sites obtained in step (7).

FIG. 6 is a standard curve chart of KIT p.D816V mutation sites obtained in step (7).

Fig. 7 is a standard graph of NPM1 p.w288cfs 12 mutation sites obtained in step (7).

FIG. 8 is a graph of PCR amplification of positive plasmids with different mutation frequency gradients.

FIG. 9 is a sequencing peak diagram of FLT3 p.D835Y mutation site with a mutation frequency of 0.1% in the PCR product obtained in step (2).

FIG. 10 is a sequencing peak diagram of FLT3 p.D835Y mutation site with a mutation frequency of 0.1% in the PCR product obtained in step (3).

FIG. 11 is a sequencing peak diagram of FLT3 p.D835Y mutation site with a mutation frequency of 0% in the PCR product obtained in step (2).

FIG. 12 is a sequencing peak diagram of FLT3 p.D835Y mutation site with a mutation frequency of 0% in the PCR product obtained in step (3).

FIG. 13 is a graph showing the sequencing peaks of the mutant site of DNMT3A p.R882H at a mutation frequency of 0.1% in the PCR product obtained in step (2).

FIG. 14 is a graph showing the sequencing peaks of the mutant site of DNMT3A p.R882H at a mutation frequency of 0.1% in the PCR product obtained in step (3).

Fig. 15 is a sequencing peak diagram of DNMT3A p.r882h mutation sites with a mutation frequency of 0% in the PCR product obtained in step (2).

FIG. 16 is a sequence peak diagram of the DNMT3A p.R882H mutation site with a mutation frequency of 0% in the PCR product obtained in step (3).

FIG. 17 is a sequence peak diagram of IDH1 p.R132H mutation site with mutation frequency of 0.1% in the PCR product obtained in step (2).

FIG. 18 is a sequence peak diagram of IDH1 p.R132H mutation site with a mutation frequency of 0.1% in the PCR product obtained in step (3).

FIG. 19 is a sequence peak diagram of IDH1 p.R132H mutation site with mutation frequency of 0% in the PCR product obtained in step (2).

FIG. 20 is a graph showing the sequencing peaks of the mutation sites of IDH1 p.R132H with the mutation frequency of 0% in the PCR product obtained in step (3).

FIG. 21 is a graph showing the sequencing peaks of the mutation sites in the PCR product obtained in step (2), wherein the mutation frequency is 0.1% IDH2 p.R140Q.

FIG. 22 is a sequence peak diagram of the mutation site of the PCR product obtained in step (3) with a mutation frequency of 0.1% IDH2 p.R140Q.

FIG. 23 is a graph showing the sequencing peaks of the mutation sites of IDH2 p.R140Q with a mutation frequency of 0% in the PCR product obtained in step (2).

FIG. 24 is a graph showing the sequencing peaks of the mutation sites of 0% IDH2 p.R140Q in the PCR product obtained in step (3).

FIG. 25 is a sequence peak diagram of the mutation site of IDH2 p.R172K with a mutation frequency of 0.1% in the PCR product obtained in step (2).

FIG. 26 is a sequence peak diagram of the mutation site of IDH2 p.R172K with the mutation frequency of 0.1% in the PCR product obtained in step (3).

FIG. 27 is a diagram showing the sequencing peaks of the mutation sites of IDH2 p.R172K with the mutation frequency of 0% in the PCR product obtained in step (2).

FIG. 28 is a diagram showing the sequencing peaks of the mutation sites of IDH2 p.R172K with the mutation frequency of 0% in the PCR product obtained in step (3).

FIG. 29 is a sequencing peak diagram of KIT p.D816V mutation site with a mutation frequency of 0.1% in the PCR product obtained in step (2).

FIG. 30 is a sequencing peak diagram of KIT p.D816V mutation site with a mutation frequency of 0.1% in the PCR product obtained in step (3).

FIG. 31 is a sequencing peak diagram of KIT p.D816V mutation site with mutation frequency of 0% in the PCR product obtained in step (2).

FIG. 32 is a sequencing peak diagram of KIT p.D816V mutation site with a mutation frequency of 0% in the PCR product obtained in step (3).

FIG. 33 is a sequence peak of the mutation site of NPM1 p.W288Cfs 12 with a mutation frequency of 0.1% in the PCR product obtained in step (2).

FIG. 34 is a sequence peak of the mutation site of NPM1 p.W288Cfs 12 with a mutation frequency of 0.1% in the PCR product obtained in step (3).

Fig. 35 is a sequencing peak of NPM1 p.w288cfs 12 mutation site with a mutation frequency of 0% in the PCR product obtained in step (2). 1-

Fig. 36 is a sequencing peak of NPM1 p.w288cfs 12 mutation site with a mutation frequency of 0% in the PCR product obtained in step (3).

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. In addition, the apparatus and reagents used in the following examples are commercially available ones unless otherwise specified.

Example 1

This example provides a primer set for detecting AML-related gene mutation, which includes FLT3 gene primer pair, DNMT3A gene primer pair, IDH1 gene primer pair, IDH2 gene first primer pair, IDH2 gene second primer pair, KIT gene primer pair, and NPM1 gene primer pair; the nucleotide sequence of the FLT3 gene primer pair is shown in a sequence table SEQ ID NO 1-2; the nucleotide sequence of the DNMT3A gene primer pair is shown as a sequence table SEQ ID NO. 4-5; the nucleotide sequence of the IDH1 gene primer pair is shown in a sequence table SEQ ID NO. 7-8; the nucleotide sequence of the IDH2 gene first primer pair is shown in a sequence table SEQ ID NO. 10-11; the nucleotide sequence of the second primer pair of the IDH2 gene is shown in a sequence table SEQ ID NO. 13-14; the nucleotide sequence of the KIT gene primer pair is shown as a sequence table SEQ ID NO 16-17; the nucleotide sequence of the NPM1 gene primer pair is shown in a sequence table SEQ ID NO. 19-20. Specifically, the primer pairs for each gene are shown in table 1 below:

TABLE 1

The primers designed by the embodiment of the invention mainly aim at hot spot mutation regions related to the AML of the 6 genes, wherein the same pair of primers and probes can detect various mutation modes. Specifically, the FLT3 gene primer pair is used for detecting point D835 and point I836 mutations of FLT3 gene, the DNMT3A gene primer pair is used for detecting point R882 mutations of DNMT3A gene, the IDH1 gene primer pair is used for detecting point R132 mutation of IDH1 gene, the IDH2 first gene primer pair and the IDH1 second gene primer pair are respectively used for detecting point R140 and point R172 mutations of IDH2 gene, the KIT gene primer pair is used for detecting point D816 mutation of KIT gene, and the NPM1 gene primer pair is used for detecting point W288fs × 12 mutation of NPM1 gene.

Example 2

The embodiment provides a kit for detecting AML-related gene mutation, which comprises a PCR amplification reaction reagent, a positive quality control product, a negative quality control product and a standard curve equation, wherein the kit further comprises the primer group and a primer probe group corresponding to the primer group. The corresponding gene primer pairs between the primer groups exist in a mixed solution mode, the probes of the primer probe group can exist in a mixed solution mode with the primer pairs of the corresponding genes, the molar concentration of each primer in the primer group is 2-100 mu mol/L, and the molar concentration of each probe in the probe group is 10-1000 mu mol/L. The probe has an overlapping sequence of 5-13bp with an upstream primer in a primer pair of a corresponding gene, 4 mismatched bases are designed at the 3' end of the probe or C3 Spacer modification is carried out, and when the probe is combined with a wild-type template, amplification of the wild-type template can be competitively inhibited.

Wherein the primer probe group comprises the primer group, an FLT3 gene probe, a DNMT3A gene probe, an IDH1 gene probe, an IDH2 gene first probe, an IDH2 gene second probe, a KIT gene probe and an NPM1 gene probe; the nucleotide sequence of the FLT3 gene probe is shown in a sequence table SEQ ID NO. 3; the nucleotide sequence of the DNMT3A gene probe is shown in a sequence table SEQ ID NO. 6; the nucleotide sequence of the IDH1 gene probe is shown in a sequence table SEQ ID NO. 9; the nucleotide sequence of the first probe of the IDH2 gene is shown in a sequence table SEQ ID NO. 12; the nucleotide sequence of the second probe of the IDH2 gene is shown in a sequence table SEQ ID NO. 15; the nucleotide sequence of the KIT gene probe is shown in a sequence table SEQ ID NO. 18; the nucleotide sequence of the NPM1 gene probe is shown in a sequence table SEQ ID NO: 21. Specifically, the sequences of the probes for the respective genes are shown in table 2 below.

TABLE 2

In addition, the PCR amplification reaction reagent provided in this example is ZoomBDA Master Mix; the negative quality control product is the whole blood genome DNA of a healthy person; the positive quality control product is a mixed solution of mutant plasmids of all genes and healthy human genome DNA; wherein the positive quality control substances comprise FLT3 gene mutation plasmids, DNMT3A gene mutation plasmids, IDH1 gene mutation plasmids, IDH2 gene first mutation plasmids, IDH2 gene second mutation plasmids, KIT gene mutation plasmids and NPM1 gene mutation plasmids. The nucleotide sequence of the FLT3 gene mutant plasmid is shown in a sequence table SEQ ID NO. 22; the nucleotide sequence of the DNMT3A gene mutant plasmid is shown in a sequence table SEQ ID NO. 23; the nucleotide sequence of the IDH1 gene mutant plasmid is shown in a sequence table SEQ ID NO. 24; the nucleotide sequence of the first mutant plasmid of the IDH2 gene is shown in a sequence table SEQ ID NO. 25; the nucleotide sequence of the second mutant plasmid of the IDH2 gene is shown in a sequence table SEQ ID NO: 26; the nucleotide sequence of the KIT gene mutant plasmid is shown as a sequence table SEQ ID NO. 18; the nucleotide sequence of the NPM1 gene mutant plasmid is shown in a sequence table SEQID NO. 21.

Example 3

This example provides a method for detecting AML-associated genetic mutation using the kit provided in example 2 above, wherein the sample should be analyzed simultaneously with the positive quality control and the negative quality control in each detection reaction. Which comprises the following steps:

(1) obtaining DNA of a sample to be detected as a template; specifically, the value of the DNA OD260/OD280 is 1.8-2.0, the concentration of the DNA is diluted to 10 ng/mu L, and the template is immediately detected or stored at-20 ℃ for later use.

(2) Mixing the primer probe set, the PCR amplification reaction reagent and the template together for PCR amplification reaction to obtain a first CT value; specifically, 6 μ L of the seven sets of primer probe mixed solutions provided in example 2, seven sets of 25 μ L of PCR amplification reaction reagents, and seven sets of 9 μ L of nuclease-free water are taken to prepare seven sets of reaction solutions, the seven sets of reaction solutions are placed in the wells corresponding to the 96-well plate or the 8-tube, 10 μ L of the template obtained in step (1) is added, and then the seven sets of reaction solutions are placed in the ABI 7500PCR instrument to perform PCR reaction, so as to obtain seven sets of first CT values.

(3) Mixing the primer group, the PCR amplification reaction reagent and the template together for PCR amplification reaction to obtain a second CT value; specifically, 4 μ L of the seven groups of primer mixed solutions, seven groups of 25 μ L of PCR amplification reaction reagents and seven groups of 11 μ L of nuclease-free water are taken to prepare seven groups of reaction solutions, the seven groups of reaction solutions are respectively placed in a 96-well plate or a hole corresponding to an 8-connecting pipe, 10 μ L of the template obtained in the step (1) is respectively added, and then the seven groups of reaction solutions are placed in an ABI 7500PCR instrument for PCR reaction to obtain seven groups of second CT values.

(4) Carrying out PCR amplification reaction on the negative quality control product to obtain a negative reference CT difference value; specifically, a negative quality control substance solution is taken to replace the template in the step (2) for PCR reaction, a negative quality control substance solution is taken to replace the template in the step (3) for PCR reaction, and CT values obtained by two groups of PCR reactions are different to obtain a negative reference CT difference value; for the mutation points of seven groups of different genes and seven groups of the probes, CT differences corresponding to the seven groups can be obtained.

(5) Carrying out PCR amplification reaction on the positive quality control product to obtain a positive reference CT difference value; specifically, a positive quality control substance solution is taken to replace the template in the step (2) for PCR reaction, a positive quality control substance solution is taken to replace the template in the step (3) for PCR reaction, and CT values obtained by two groups of PCR reactions are different to obtain a positive reference CT difference value; for the mutation points of seven groups of different genes and seven groups of the probes, CT differences corresponding to the seven groups can be obtained.

(6) Subtracting the seven groups of first CT values from the seven groups of second CT values of the corresponding genes respectively to obtain seven groups of CT difference values; and comparing the seven groups of CT difference values with the reference CT difference values of the mutation points of the seven groups of corresponding genes respectively, and judging whether the genes corresponding to the primer groups have mutation. And if the CT difference value is between the negative reference CT difference value and the positive reference CT difference value, the detection result of the sample is considered to be positive or weakly positive, and corresponding gene mutation may exist.

(7) Respectively carrying out Sanger sequencing on the PCR products obtained in the steps (2) and (3) to obtain sequencing peak diagrams; specifically, the products from the probed and non-probed reaction tubes of each sample were collected separately, one collection tube for each well, and Sanger sequencing was performed by sequencing company, and all genes were sequenced using M13RP primer. Sequence alignment analysis was performed using software such as sequenci nganalysis, Bioedit, Snapgene, etc. And comparing the sequence obtained by sequencing with the detection gene amplification sequence, and identifying the gene mutation type.

(8) And substituting the difference value of the first CT value and the second CT value into a standard curve equation corresponding to the mutation type according to the identified mutation type to obtain the mutation frequency of the gene corresponding to the primer group. Specifically, the difference value between the first CT value and the second CT value is taken as a Y value and is brought into a standard curve equation, the obtained X is a logarithm value (log (VAF)) of the mutation frequency of the corresponding BRAF gene mutation type, and then 10 is calculated^XNamely the mutation frequency of the mutation type of the BRAF gene to be detected. The standard curve graphs and equations of the seven groups of different gene mutation points are respectively shown in fig. 1-7, and it can be seen that the standard curves of the mutant types of different genes are different, so that the standard curve equations corresponding to the mutant types are required to be accurately calculated when the gene mutation frequency is quantitatively calculated.

In addition, one set of the mutant plasmids and the healthy human genome DNA were mixed at mutation frequencies of 0%, 0.1%, 1%, 5%, 10%, and 50%, respectively, and PCR reactions were performed according to the steps (2) and (3), and 6 sets of PCR amplification curves were obtained as shown in FIG. 8, in which A to F correspond to concentrations of 0%, 0.1%, 1%, 5%, 10%, and 50%, respectively. As can be seen from the figure, the sensitivity of the method for detecting AML-associated genetic mutations provided in the examples of the present invention was 0.1%. The qPCR products at 0.1% and 0% of each gene were Sanger sequenced and the sequencing peaks for each gene are shown in figures 9-36. Wherein FIGS. 9 to 12 are the sequencing peak diagrams of the primer probe set products and the primer set products with the mutation frequencies of FLT3 gene of 0.1% and 0%, respectively. As can be seen from the figure, when the mutation frequency is 0.1%, in the reaction in which the probe is added, there is a clear overlap peak at the mutation position; the reaction without the probe is added, and the sequencing result is a single peak and is a wild type; the result shows a wild-type single peak for the mutation frequency of 0%, regardless of the probe-added or non-probe-added sequencing peak pattern.

FIGS. 13 to 16 are the results of the primer probe set products and the primer set products having the mutation frequencies of DNMT3A gene of 0.1% and 0%, respectively. As can be seen from the figure, when the mutation frequency is 0.1%, in the reaction in which the probe is added, there is a single mutant peak at the mutation position; but not the reaction with the addition of probe, resulting in a wild type single peak; the result of the sequencing peak pattern of the primer probe set or the primer set is shown as a wild-type single peak for the mutation frequency of 0%, namely, the result of the negative sample.

FIGS. 17 to 20 are sequencing peak diagrams of primer probe set products and primer set products with IDH1 gene mutation frequencies of 0.1% and 0%, respectively. As can be seen from the figure, when the mutation frequency was 0.1%, the result of the primer probe set showed a clear set of peaks at the mutation positions; the primer group shows a wild type single peak and has no mutation; when the mutation frequency was 0%, the sequencing peak pattern of either the primer probe set or the primer set showed a wild-type single peak.

FIGS. 21 to 24 are the results of the primer probe set products and the primer set products with the frequency of mutation at the R140 site of IDH2 gene of 0.1% and 0%, respectively. As can be seen from the figure, at a mutation frequency of 0.1%, there was a significant set of peaks at the mutation position in the reaction of the primer probe set, while the wild-type single peak was shown in the reaction of the primer set; when the mutation frequency was 0%, the result showed a wild type single peak regardless of the sequencing peak pattern of the primer probe set or the sequencing peak pattern of the primer set.

FIGS. 25 to 28 are the results of the primer probe set products and the primer set products with the frequency of mutation at the R172 site of IDH2 gene of 0.1% and 0%, respectively. As can be seen from the figure, at a mutation frequency of 0.1%, there was a clear set of peaks at the mutation positions in the reaction of the primer probe set, while the wild type was shown on the peak map in the reaction of the primer set; when the mutation frequency was 0%, the result showed a wild type single peak regardless of the sequencing peak pattern of the primer probe set or the sequencing peak pattern of the primer set.

FIGS. 29-32 are the results of primer probe set products and primer set products with mutation frequencies of KIT gene of 0.1% and 0%, respectively. As can be seen from the figure, when the mutation frequency is 0.1%, the reaction added with the probe has a clear set of peaks at the mutation site, while the reaction without the probe shows a wild type single peak without mutation; when the mutation frequency is 0%, the result is a wild-type single peak regardless of the sequencing peak pattern with the probe or the sequencing peak pattern without the probe.

FIGS. 33 to 36 are the results of the primer probe set products and the primer set products with the NPM1 gene mutation frequencies of 0.1% and 0%, respectively. As can be seen from the figure, when the mutation frequency is 0.1%, in the reaction in which the probe is added, there are clear double peaks just after the mutation site, and the main peak is the mutant sequence; and the reaction without the probe is shown as a wild type single peak; when the mutation frequency is 0%, the result is a wild-type single peak regardless of the sequencing peak pattern with the probe or the sequencing peak pattern without the probe.

As can be seen from the sequencing peak diagrams of the 0.1% standard products of the different genes, after the qPCR product in the primer probe set is sequenced by Sanger, the wild type and the mutant type exist at the same time, and the peak diagrams are clear; the primer group is only shown as a wild type, which indicates that when the mutation frequency is 0.1%, a large amount of mutant sequences with a small number of molecules are enriched under the action of the primer probe and can be detected by Sanger; meanwhile, by combining a 0% sequencing result, the low mutation frequency of 0.1% and the standard substance of 0% (wild type) can be clearly distinguished, and the product is single and has no impurity peak, so that the primer and the probe have higher specificity, and the method for detecting AML related gene mutation provided by the embodiment of the invention has higher sensitivity and specificity.

When the ABI 7500PCR instrument is used, the "SYBR Green Reagents" mode is selected, and the PCR program shown in table 3 is set:

TABLE 3

In light of the foregoing description of the preferred embodiment of the present invention, many modifications and variations will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.

Sequence listing

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<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

agcggataac aatttcacac aggaactcca ggataataca catcacagt 49

<210>3

<211>33

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

tggaatcact catgatatct cgagccattt ttt 33

<210>4

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

agcagtctct gcctcgc 17

<210>5

<211>47

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

agcggataac aatttcacac aggaagaaga ttcggcagaa ctaagca 47

<210>6

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

cctcgccaag cggctcatgt taatatt 27

<210>7

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

gcttgtgagt ggatgggtaa aac 23

<210>8

<211>46

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

agcggataac aatttcacac aggatgtgtt gagatggacg cctatt 46

<210>9

<211>36

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

tgggtaaaac ctatcatcat aggtcgtcat gaaaaa 36

<210>10

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

agaagatgtg gaaaagtccc aatg 24

<210>11

<211>42

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

agcggataac aatttcacac aggagtgccc aggtcagtgg at 42

<210>12

<211>31

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

tcccaatgga actatccgga acatccaaaa a 31

<210>13

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

ctggtcgcca tgggcgt 17

<210>14

<211>48

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>14

agcggataac aatttcacac aggatgaaga agatgtggaa aagtccca 48

<210>15

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>15

ggcgtgcctg ccaatggtga attaa 25

<210>16

<211>32

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>16

tcctttaacc acataattag aatcattctt ga 32

<210>17

<211>56

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>17

agcggataac aatttcacac aggaagttag ttttcactct ttacaagtta aaatga 56

<210>18

<211>30

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>18

atcattcttg atgtctctgg ctagaccaaa 30

<210>19

<211>34

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>19

tttctttttt tttttttcca ggctattcaa gatc 34

<210>20

<211>48

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>20

agcggataac aatttcacac aggacctgga caacatttat caaacacg 48

<210>21

<211>34

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>21

gctattcaag atctctggca gtggaggaaa aaaa 34

<210>22

<211>500

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>22

tcactttgtt tgttgcacat catcatggcc gctcacggca cagcccagta aagataagag 60

gccttccatc accggtacct cctactgaag ttgagtctag aagaaagatt gcactccagg 120

ataatacaca tcacagtaaa taacactctg gtgtcattct tgacagtgtg ttcacagaga 180

cctggccgcc aggaacgtgc ttgtcaccca cgggaaagtg gtgaagatat gtgactttgg 240

attggctcga tatatcatga gtgattccaa ctatgttgtc aggggcaatg tgaggctgct 300

atttcctact tatttttata cggctatttt gtgttgtgtc gttatcatgg taaacaactg 360

cactcactgt ggtgcatttt tgatttatgg taacatcaaa aaaccctcac agcagtctgc 420

ttacttatgc ttaaaaggtt tttctgcagc ttcagggaat cttccatcta taataaaagc 480

tgagcaaaat agcatcacat 500

<210>23

<211>500

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>23

tgatctgagt gccgggttgt ttataaagga cagaagattc ggcagaacta agcaggcgtc 60

agaggagttg gtgggtgtga gtgcccctgt ccctgcactt cgggtggctg ctggtcctcc 120

gggtcctgct gtgtggttag acggcttccg ggcagcctgg tctggccagc actcaccctg 180

ccctctctgc cttttctccc ccagggtatt tggtttccca gtccactata ctgacgtctc 240

caacatgagc cacttggcga ggcagagact gctgggccgg tcatggagcg tgccagtcat 300

ccgccacctc ttcgctccgc tgaaggagta ttttgcgtgt gtgtaaggga catgggggca 360

aactgaggta gcgacacaaa gttaaacaaa caaacaaaaa acacaaaaca taataaaaca 420

ccaagaacat gaggatggag agaagtatca gcacccagaa gagaaaaagg aatttaaaac 480

aaaaaccaca gaggcggaaa 500

<210>25

<211>500

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>25

tcgtgatgcc accaacgacc aagtcaccaa ggatgctgca gaagctataa agaagcataa 60

tgttggcgtc aaatgtgcca ctatcactcc tgatgagaag agggttgagg agttcaagtt 120

gaaacaaatg tggaaatcac caaatggcac catacgaaat attctgggtg gcacggtctt 180

cagagaagcc attatctgca aaaatatccc ccggcttgtg agtggatggg taaaacctat 240

catcataggt catcatgctt atggggatca agtaagtcat gttggcaata atgtgatttt 300

gcatgttttt tttttcatgg cccagaaatt tccaacttgt atgtgtttta ttcttatctt 360

ttggtatcta cacccattaa gcaaggtatg aaattgagaa atgcatatat gtataactgt 420

atatttacac acatttagct aaaggcaaat acaaataaac ttacaaatag gcgtccatct 480

caacacattt tttttcaaac 500

<210>25

<211>500

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>25

cagactccag agcccacaca tttgcactct agactctact gccttcctca tgaagaattt 60

taggaccccc gtctggctgt gttgttgctt ggggttcaaa ttctggttga aagatggcgg 120

ctgcagtggg accactatta tctctgtcct cacagagttc aagctgaaga agatgtggaa 180

aagtcccaat ggaactatcc agaacatcct gggggggact gtcttccggg agcccatcat 240

ctgcaaaaac atcccacgcc tagtccctgg ctggaccaag cccatcacca ttggcaggca 300

cgcccatggc gaccaggtag gccagggtgg agaggggatc cactgacctg ggcacccccc 360

gactggagct cctcgcctag ccatcctctt gtctctgcag tacaaggcca cagactttgt 420

ggcagaccgg gccggcactt tcaaaatggt cttcacccca aaagatggca gtggtgtcaa 480

ggagtgggaa gtgtacaact 500

<210>26

<211>500

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>26

cagactccag agcccacaca tttgcactct agactctact gccttcctca tgaagaattt 60

taggaccccc gtctggctgt gttgttgctt ggggttcaaa ttctggttga aagatggcgg 120

ctgcagtggg accactatta tctctgtcct cacagagttc aagctgaaga agatgtggaa 180

aagtcccaat ggaactatcc ggaacatcct gggggggact gtcttccggg agcccatcat 240

ctgcaaaaac atcccacgcc tagtccctgg ctggaccaag cccatcacca ttggcaagca 300

cgcccatggc gaccaggtag gccagggtgg agaggggatc cactgacctg ggcacccccc 360

gactggagct cctcgcctag ccatcctctt gtctctgcag tacaaggcca cagactttgt 420

ggcagaccgg gccggcactt tcaaaatggt cttcacccca aaagatggca gtggtgtcaa 480

ggagtgggaa gtgtacaact 500

<210>27

<211>500

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>27

ttcaaggcgt acttttgatc cttattcttg gtgtactgaa tactttaaaa caaaagtatt 60

ggatttttta taatataagc aacactatag tattaaaaag ttagttttca ctctttacaa 120

gttaaaatga atttaaatgg ttttcttttc tcctccaacc taatagtgta ttcacagaga 180

cttggcagcc agaaatatcc tccttactca tggtcggatc acaaagattt gtgattttgg 240

tctagccaga gtcatcaaga atgattctaa ttatgtggtt aaaggaaacg tgagtaccca 300

ttctctgctt gacagtcctg caaaggattt ttagtttcaa ctttcgataa aaattgtttc 360

ctgtgatttt cataatgtaa atcctgtcta gggatatcac acattttagc agtcaaatta 420

agtatacttc agcaaaattt gcatggtatg ctgaacatta ctacaactaa cattcaataa 480

tagaagtcct aattctaatt 500

<210>28

<211>500

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>28

aatgctaaac tattaaataa ttattagtat attgttcaca tttttatgac tgattaaagt 60

gtttggaatt aaattacatc tgagtataaa ttttcttgga gtcatatctt tatctagagt 120

taactctctg gtggtagaat gaaaaataga tgttgaacta tgcaaagaga catttaattt 180

attgatgtct atgaagtgtt gtggttcctt aaccacattt cttttttttt ttttccaggc 240

tattcaagat ctctgtctgg cagtggagga agtctcttta agaaaatagt ttaaacaatt 300

tgttaaaaaa ttttccgtct tatttcattt ctgtaacagt tgatatctgg ctgtcctttt 360

tataatgcag agtgagaact ttccctaccg tgtttgataa atgttgtcca ggttctattg 420

ccaagaatgt gttgtccaaa atgcctgttt agtttttaaa gatggaactc caccctttgc 480

ttggttttaa gtatgtatgg 500

Claims (10)

1. The primer group for detecting AML related gene mutation is characterized by comprising one or more of FLT3 gene primer pair, DNMT3A gene primer pair, IDH1 gene primer pair, IDH2 gene first primer pair, IDH2 gene second primer pair, KIT gene primer pair and NPM1 gene primer pair; the nucleotide sequence of the FLT3 gene primer pair is shown in a sequence table SEQ ID NO. 1-2; the nucleotide sequence of the DNMT3A gene primer pair is shown as a sequence table SEQ ID NO. 4-5; the nucleotide sequence of the IDH1 gene primer pair is shown in a sequence table SEQ ID NO. 7-8; the nucleotide sequence of the IDH2 gene first primer pair is shown in a sequence table SEQ ID NO. 10-11; the nucleotide sequence of the second primer pair of the IDH2 gene is shown in a sequence table SEQ ID NO. 13-14; the nucleotide sequence of the KIT gene primer pair is shown as a sequence table SEQ ID NO 16-17; the nucleotide sequence of the NPM1 gene primer pair is shown in a sequence table SEQ ID NO. 19-20.

2. A kit for detecting AML-associated gene mutation, comprising PCR amplification reaction reagents, positive quality control substances, negative quality control substances and a standard curve equation, wherein the kit further comprises the primer set according to claim 1 and a primer probe set corresponding to the primer set.

3. The KIT for detecting AML-associated genetic mutation according to claim 2 wherein said primer probe set comprises the primer set of claim 1 and one or more of FLT3 gene probe, DNMT3A gene probe, IDH1 gene probe, IDH2 gene first probe, IDH2 gene second probe, KIT gene probe and NPM1 gene probe; the nucleotide sequence of the FLT3 gene probe is shown in a sequence table SEQ ID NO. 3; the nucleotide sequence of the DNMT3A gene probe is shown in a sequence table SEQ ID NO. 6; the nucleotide sequence of the IDH1 gene probe is shown in a sequence table SEQ ID NO. 9; the nucleotide sequence of the first probe of the IDH2 gene is shown in a sequence table SEQ ID NO. 12; the nucleotide sequence of the second probe of the IDH2 gene is shown in a sequence table SEQ ID NO. 15; the nucleotide sequence of the KIT gene probe is shown in a sequence table SEQ ID NO. 18; the nucleotide sequence of the NPM1 gene probe is shown in a sequence table SEQ ID NO: 21.

4. The kit for detecting AML-associated gene mutation according to claim 2 wherein the molar concentration of each primer in the primer set is 2-100. mu. mol/L.

5. The kit for detecting AML-associated genetic mutation according to claim 2 wherein the primer probe set has a molar concentration of 2-100. mu. mol/L for each primer and 10-1000. mu. mol/L for each probe.

6. The KIT for detecting AML-associated gene mutation according to claim 2 wherein said positive quality control substances comprise one or more of FLT3 gene mutation plasmid, DNMT3A gene mutation plasmid, IDH1 gene mutation plasmid, IDH2 gene first mutation plasmid, IDH2 gene second mutation plasmid, KIT gene mutation plasmid and NPM1 gene mutation plasmid.

7. The kit for detecting AML-associated genetic mutation according to claim 6, wherein the nucleotide sequence of FLT3 gene mutant plasmid is shown in sequence Listing SEQ ID NO: 22; the nucleotide sequence of the DNMT3A gene mutant plasmid is shown in a sequence table SEQ ID NO. 23; the nucleotide sequence of the IDH1 gene mutant plasmid is shown in a sequence table SEQ ID NO. 24; the nucleotide sequence of the first mutant plasmid of the IDH2 gene is shown in a sequence table SEQ ID NO. 25; the nucleotide sequence of the second mutant plasmid of the IDH2 gene is shown in a sequence table SEQ ID NO: 26; the nucleotide sequence of the KIT gene mutant plasmid is shown as a sequence table SEQ ID NO. 18; the nucleotide sequence of the NPM1 gene mutant plasmid is shown in a sequence table SEQ ID NO: 21.

8. The kit for detecting a BRAF gene mutation according to claim 2, wherein the standard curve equation is a standard curve equation obtained by subtracting the first CT value and the second CT value of the standard from the mutation plasmid and the healthy human genomic DNA at a mutation frequency of 50%, 10%, 5%, 1%, 0.1% and 0%, and specifically comprises the following steps:

mixing the primer probe set according to claim 2 or 3 or 5, the PCR amplification reaction reagent according to claim 2 and the 6 mutation frequency standards together to perform PCR amplification reaction, so as to obtain first CT values of the 6 mutation frequency standards;

mixing the primer set according to claim 2 or 4, the PCR amplification reaction reagent according to claim 2 and 6 mutation frequency standards together to perform PCR amplification reaction, so as to obtain second CT values of the 6 mutation frequency standards;

and obtaining a standard curve equation according to the difference value of the first CT value and the second CT value of the 6 standard products.

9. A method for detecting mutation in AML-associated gene comprising the steps of:

obtaining DNA of a sample to be detected as a template;

mixing the primer probe set according to claim 2 or 3 or 5, the PCR amplification reaction reagent according to claim 2 and the template for PCR amplification reaction to obtain a first CT value;

mixing the primer set according to claim 2 or 4, the PCR amplification reaction reagent according to claim 2 and the template together for PCR amplification reaction to obtain a second CT value;

carrying out PCR amplification reaction on the negative quality control material according to claim 2 to obtain a negative reference CT difference value;

performing PCR amplification reaction on the positive quality control product according to claim 2 to obtain a positive reference CT difference value;

and comparing the difference value of the first CT value and the second CT value with the positive reference CT difference value and the negative reference CT difference value, and judging whether the gene corresponding to the primer group has mutation.

10. The method for detecting AML-related genetic mutation as defined in claim 9, further comprising:

sequencing the PCR product obtained by the amplification reaction to obtain a sequencing result;

analyzing the sequencing result, and identifying the gene mutation type related to AML;

and substituting the difference value of the first CT value and the second CT value into a standard curve equation corresponding to the mutation type according to the identified mutation type to obtain the mutation frequency of the gene corresponding to the primer group.

Images