CN111154881A

CN111154881A - Detection kit for gene mutation in acute myeloid leukemia and application

Info

Publication number: CN111154881A
Application number: CN202010156293.8A
Authority: CN
Inventors: 唐春花; 邢宽; 谢珍; 张鹏; 袁鸣; 安雪茄; 高婷婷; 高彧辉
Original assignee: Nanjing Experimental Medicine Examines Co ltd
Current assignee: Nanjing Experimental Medicine Examines Co ltd
Priority date: 2020-03-09
Filing date: 2020-03-09
Publication date: 2020-05-15

Abstract

The invention discloses a detection kit for gene mutation in acute myeloid leukemia and application thereof, wherein the application comprises adding a plurality of pairs of primers in the same PCR reaction system, each pair of primers are respectively combined at corresponding positions of a template, simultaneously amplifying a plurality of nucleic acid fragments, introducing a linker sequence (index) for high-throughput sequencing to two sides of the nucleic acid fragments through PCR reaction, and finally obtaining a nucleic acid library for high-throughput sequencing and signaling process analysis to obtain sequence information of a target region. The genetic variation condition in AML is screened by utilizing the multiplex PCR targeted high-throughput sequencing technology with high accuracy, high coverage and high detection rate, an accurate and authoritative medical interpretation report is given, and guidance is provided for disease diagnosis, prognosis stratification and targeted treatment of AML patients.

Description

Detection kit for gene mutation in acute myeloid leukemia and application

Technical Field

The invention relates to the technical field of biomedical detection, in particular to a detection kit for gene mutation in acute myeloid leukemia and application thereof.

Background

Leukemia is caused by abnormal cell proliferation, differentiation or apoptosis due to abnormal genes of hematopoietic stem cells or precursor cells, is a malignant blood disease with high heterogeneity, and the diagnosis and treatment of leukemia require comprehensive analysis from morphology, immunology, cytogenetics and molecular biology. The most common molecular biological abnormalities in leukemia mainly include: gene mutation, fusion gene and gene expression, wherein the gene mutation mainly comprises point mutation, gene fragment insertion or deletion.

Acute Myeloid Leukemia (AML) is a hematological malignancy common in children and adults. 1-2 individuals occur in every 10 thousands of people in China, mainly occur in adults, and the disease condition is acute and serious, and the prognosis is extremely dangerous. With the continuous development of molecular biology sequencing technology, a large number of molecular biological markers closely related to acute myeloid leukemia are discovered. From the NCCN guide of 2006 edition, FLT3-ITD is only used as a detection index recommended by a clinician, from the NCCN guide of 2010 edition, c-KIT, FLT3-ITD, NPM and CEBPA are definitely indicated, and from the NCCN guide of 2018 edition, IDH1, IDH2, TP53 and the like are newly added, so that clinical significance brought by gene mutation is found in a well-known and well-researched way. In 2016, WHO revised the classification of hematological tumors again, in which the subtypes of AML with cepbpa biallelic mutation and AML with NPM1 genetic mutation were defined in Acute Myeloid Leukemia (AML), and RUNX1 genetic mutation with AML was newly added, while the AML with RUNX1 mutation represented a biologically specific subtype, suggesting a possibly worse prognosis compared to other subtypes of AML. The application value of the detection of gene mutation in the diagnosis and treatment of the blood tumor is indicated in the domestic expert consensus of the 'application of the second generation sequencing technology in the blood tumor-the Chinese expert consensus' in 2018, and the application value can provide references for clinical diagnosis and typing, prognosis judgment, treatment guidance, tiny residual lesion (MRD) monitoring and clone evolution.

At present, the methods for detecting gene mutation in molecular biology mainly include generation sequencing (Sanger), high-throughput sequencing technology (NGS), real-time fluorescence PCR (RQ-PCR), digital PCR, etc., and the mainstream of the high-throughput targeted sequencing technology mainly includes targeted sequencing by multiplex PCR and targeted sequencing by capture probes. Compared with a first generation (Sanger) sequencing technology, the multiple PCR targeted sequencing technology has higher sensitivity and can quantitatively detect the gene mutation load rate; compared with the target sequencing of a capture probe, the construction process of the target sequencing technology library of the multiplex PCR is simpler, a more economical and rapid sequencing scheme can be provided, most of real-time fluorescence PCR (RQ-PCR) and digital PCR are used for detecting known mutation sites in genes, the cost of a primer probe in the experimental process is higher, and the feasibility of screening the polygenic mutation is poorer.

Therefore, the multiple PCR targeted high-throughput sequencing technology is used for screening the genetic variation condition in AML with high accuracy, high coverage and high detection rate, an accurate and authoritative medical interpretation report is given, and the problem of providing guidance for disease diagnosis, prognosis stratification and targeted treatment of AML patients is urgently to be solved.

Disclosure of Invention

The invention aims to provide a detection kit for multiplex PCR targeted high-throughput sequencing of 23 different variation types (point mutation, small fragment insertion/deletion and other mutation types) in gene coding regions with definite clinical significance in AML and application of the detection kit.

In order to achieve the above object, in a first aspect, the present invention provides a detection kit for gene mutation in acute myeloid leukemia, including primer working solution of 23 genes, a multiplex PCR reagent, absolute ethanol, purified magnetic beads and nucleic-Free Water, where the multiplex PCR reagent includes PCR mixed solution, PCR reaction solution, digestion solution, a linker P5 and a linker P7 in a multiplex amplification system, the PCR mixed solution includes Tris-HCl, 10 × buffer, 4 dNTP mixtures, and MgCl2 having a pH of 8.3, the PCR reaction solution includes Taq DNA polymerase, and the digestion solution includes shrimp alkalase and exonuclease.

Wherein the concentration of the primer working solution of the 23 genes is 4.0-6.0 pmol/ul, the concentration of the absolute ethyl alcohol is 500ml, the concentration of the purified magnetic beads is 60ml, and the concentration of the nucleic-Free Water is 5 ml.

Wherein Tris-HCl is 100mM, Taq DNA polymerase is 3000U/ml, and shrimp alkali enzyme and exonuclease are 5000U/ml.

Wherein the joint P5 is a universal primer sequence; the adaptor P7 is a base sequence for identifying a sample.

In a second aspect, the present invention provides an application of a detection kit for gene mutation in acute myeloid leukemia, comprising:

extracting DNA from a sample to be detected by using a DNA extraction kit, wherein the concentration of the extracted DNA is more than or equal to 10 ng/ul;

performing multiple PCR reaction on the extracted DNA by using a detection kit to obtain amplicon products of a plurality of target nucleotides;

purifying the amplicon products of the plurality of target nucleotides to obtain pure amplicon products;

adding a joint P5, a joint P7, a PCR mixed solution and a PCR reaction solution into a purified amplicon product serving as a template to perform PCR reaction to obtain a new amplicon library;

adding purified magnetic beads to combine with the target fragments to recover amplicon products, and washing to remove reaction impurities to obtain a pure amplicon library;

and (3) carrying out high-throughput sequencing and signal-generating flow analysis on the pure amplicon library to obtain the sequence information of the target region, and obtaining a gene mutation result.

In one embodiment, a DNA extraction kit is used to extract DNA from a sample to be tested, wherein the extracted DNA has a concentration greater than or equal to 10ng/ul, and specifically comprises:

25ng of DNA, 5ul of PCR mixed solution, 1ul of PCR reaction solution, 1ul of upstream primer and 1ul of downstream primer are complemented to 10ul by a nucleic-Free Water system, the PCR program is operated to pre-denature for 15min at 95 ℃, and then 24 cycles of 30s at 95 ℃, 90s at 60 ℃ and 90s at 72 ℃ are carried out, 10min at 72 ℃ and 4 ℃ are temporarily stored, so that a plurality of target nucleotide amplicon products are obtained.

In one embodiment, the purification of the amplicon products of the plurality of target nucleotides to obtain a purified amplicon product comprises:

adding digestive juice, running the program on a PCR instrument at 37 ℃ for 30min and at 80 ℃ for 10min, adding purified magnetic beads to combine with the target fragments to recover amplicon products, and washing to remove reaction impurities to obtain pure amplicon products.

The invention relates to a detection kit for gene mutation in acute myeloid leukemia and application thereof, wherein a plurality of pairs of primers are added in the same PCR reaction system, each pair of primers are respectively combined at corresponding positions of a template, a plurality of nucleic acid fragments are simultaneously amplified, a linker sequence (index) for high-throughput sequencing is introduced to two sides of the nucleic acid fragments through PCR reaction, and finally a nucleic acid library is obtained to be subjected to high-throughput sequencing and signaling flow analysis to obtain sequence information of a target region. The genetic variation condition in AML is screened by utilizing the multiplex PCR targeted high-throughput sequencing technology with high accuracy, high coverage and high detection rate, an accurate and authoritative medical interpretation report is given, and guidance is provided for disease diagnosis, prognosis stratification and targeted treatment of AML patients.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a schematic diagram of the multiplex PCR targeted sequencing detection technique of the present invention;

FIG. 2 is a graph showing the results of library detection by the multiplex PCR detection technique according to the present invention;

FIG. 3 is a first generation sequencing verification peak diagram of the mutation sites according to the present invention, wherein the specific gene sites are: NPM1(NM — 001355006) c.863_864insTCTG p.w288fs 12 (hybrid);

FIG. 4 is a schematic flow chart showing the application of the detection kit for gene mutation in acute myeloid leukemia according to the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

In a first aspect, the invention provides a detection kit for gene mutation in acute myeloid leukemia, which comprises primer working solution of 23 genes, a multiplex PCR reagent, absolute ethyl alcohol, purified magnetic beads and nucleic-Free Water, wherein the multiplex PCR reagent comprises PCR mixed solution, PCR reaction solution, digestion solution, a joint P5 and a joint P7 in a multiplex amplification system, the PCR mixed solution comprises Tris-HCl, 10 xbuffer, 4 dNTP mixtures and MgCl2 with the pH value of 8.3, the PCR reaction solution comprises Taq DNA polymerase, and the digestion solution comprises shrimp alkalase and exonuclease. The 23 genes are: ASXL1, BCOR, CEBPA, DNMT3A, EZH2, FLT3, IDH1, IDH2, KIT, KMT2A, KRAS, NPM1, NRAS, PHF6, RUNX1, SF3B1, SRSF2, STAG2, TET2, TP53, U2AF1, WT1, ZRSR 2. The concentration of the primer working solution of the 23 genes is 4.0-6.0 pmol/ul, the concentration of the absolute ethyl alcohol is 500ml, the concentration of the purified magnetic beads is 60ml, and the concentration of the nucleic-Free Water is 5 ml. The Tris-HCl is 100mM, the Taq DNA polymerase is 3000U/ml, and the shrimp alkali enzyme and the exonuclease are 5000U/ml. The joint P5 is a universal primer sequence; the adaptor P7 is a base sequence for identifying a sample. The multiplex PCR reagent is 96 parts.

Aiming at the defects in the existing gene mutation detection technology, the invention designs a primer sequence for detecting a plurality of exons of 23 genes of Acute Myeloid Leukemia (AML) based on the latest version of WHO 2016 edition of guide, NCCN 2019 edition of guide, blood tumor Chinese expert consensus and blood tumor related gene database. The application has the advantages of high accuracy, high sensitivity and high flux, and can quickly and efficiently carry out standardized quantitative detection on 23 Acute Myeloid Leukemia (AML) genes.

The invention arranges 23 genes related to Acute Myeloid Leukemia (AML) based on 2016 updated WHO blood tumor classification standard, NCCN 2019 guide, 2018 blood tumor Chinese expert consensus and related authority documents, and designs a multi-primer by performing sequence comparison analysis on the 23 genes in database Genbank. The invention relates to classical transcripts of 23 genes related to Acute Myeloid Leukemia (AML) as follows: ASXL1(NM _015338.5), BCOR (NM _017745.5), CEBPA (NM _004364.4), DNMT3A (NM _022552.4), EZH2(NM _004456.4), FLT3(NM _004119.2), IDH1(NM _005896.3), IDH2(NM _002168.3), KIT (NM _000222.2), KMT2A (NM _001197104.1), KRAS (NM _033360.3), NPM 033360.3 (NM _033360.3), NRAS (RSR _033360.3), PHF 033360.3 (NM _033360.3), RUNX 033360.3 (NM _033360.3), SF3B 033360.3 (NM _033360.3), SRSF 033360.3 (NM _033360.3), STAG 033360.3 (NM _033360.3), TET 033360.3 (NM _033360.3), U2AF 033360.3 (WT _033360.3), NM _033360.3 (NM _033360.3), and ZTP _ 033360.3). The designed primer Tm value is 60 +/-2 ℃, and the designed primer is reasonably distributed into a test tube after being subjected to optimization test, so that the specificity of the primer and the uniformity of amplified fragments are ensured, and the proportion of non-target fragments is reduced.

Based on the primer design principle, the 23 gene exons related to Acute Myeloid Leukemia (AML) designed by the invention particularly focus on the variation conditions of the following hot spot regions, and the corresponding amplicons are as follows:

based on the hot spot amplification region, the kit of the invention focuses on 76 hot spot mutation sites, and the correspondence between the 76 hot spot mutations and the 23 genes is shown in the following table:

the amplicon primers are designed according to the gene exons, and because Novaseq 6000 of a high-throughput sequencing illumina platform is selected for sequencing, and the adopted sequencing strategy is PE150, the sizes of target regions are all nucleotide fragments of about 300 bp.

Based on the principle of primer design, 23 kinds of gene exon primers related to Acute Myeloid Leukemia (AML) are designed by using related primer design software, and the corresponding primer sequences are as follows:

the key factors affecting the multiplex PCR primer in the invention have the following aspects:

specificity: if the specificity is not properly treated, amplification of non-target fragments can be caused, and failure of amplification of target fragments can be seriously influenced. The more the number of primers in a reaction system, the more the possibility of non-specific amplification. In the multiplex PCR experiment, it is necessary to ensure that only the target fragment is amplified and the other nucleotide fragments are not amplified.

Dimer: the generation of dimers in the amplification process can inhibit the primers from amplifying the target fragments, so that the amplification yield of the target fragments is reduced and even the amplification fails. The larger the number of primers in the same reaction system, the greater the probability of the occurrence of dimers.

Uniformity: the condition that the primer property is uniform is reflected, in addition to the factors, the multiple PCR system needs to ensure that the primer property is uniform as much as possible, and all primers have the same or similar thermodynamic stability and the like in the same system.

Therefore, the invention is finally formed by carrying out multiple testing adjustments in order to ensure that the primers in the reaction system can be effectively amplified in the same reaction environment.

When the kit is used for performing multiple PCR targeted sequencing, negative control of nucleic-Free Water is performed at the same time, so that the experimental process is not polluted, and the accuracy and reliability of data results are ensured.

The kit disclosed by the invention is operated on a Novaseq 6000 instrument using an illumia platform for detection, an instrument used in a first-generation sequencing contrast verification experiment is ABI 3730XL, and PCR reactions of the two detection technologies are operated on BIO-RAD T100.

In a second aspect, the present invention provides an application of a detection kit for gene mutation in acute myeloid leukemia, referring to fig. 4, the application of the detection kit for gene mutation in acute myeloid leukemia may include the following steps:

s101, extracting DNA from a sample to be detected by using a DNA extraction kit;

in the present embodiment, FIG. 2 is a diagram showing the library detection result of the multiplex PCR detection technique according to the present invention; FIG. 3 is a first generation sequencing verification peak diagram of the mutation sites according to the present invention, wherein the specific gene sites are: NPM1(NM — 001355006) c.863_864insTCTG p.w288fs 12 (hybrid); DNA is extracted from a sample to be detected by using the DNA extraction kit, the quality inspection ensures that the concentration of the DNA reaches more than 10ng/ul, and an electrophoresis strip has no obvious tailing so as to ensure the integrity of the DNA.

S102, performing multiple PCR reaction on the extracted DNA by using a detection kit to obtain amplicon products of a plurality of target nucleotides;

in the embodiment of the invention, FIG. 1 is a schematic diagram of the multiplex PCR targeted sequencing detection technology of the invention; the method comprises the steps of complementing 25ng of DNA, 5ul of PCR mixed solution, 1ul of PCR reaction solution, 1ul of upstream primer and 1ul of downstream primer to 10ul by using a nucleic-FreeWater, performing pre-denaturation by running a PCR program for 15min at 95 ℃, performing 24 cycles of 95 ℃ 30s, 60 ℃ 90s and 72 ℃ 90s, temporarily storing at 72 ℃ 10min and 4 ℃, and leading the 5' ends of the upstream primers to be provided with a section of the same universal sequence T1; the 5' ends of the downstream primers are all provided with another identical universal sequence T2. The T1 sequence is typically part of the 3' end of the P5 linker sequence; the T2 sequence is part of the 3' end of the P7 linker sequence. Therefore, by the round of multiplex PCR reaction, not only can the amplicon products of a plurality of target nucleotides be obtained, but also the universal sequences T1 and T2 are respectively and successfully introduced to the two sides of the amplicon products, so that preparation is made for introducing a complete adaptor sequence for the subsequent PCR reaction, namely the 1 st round of multiplex PCR reaction.

S103, purifying the amplicon products of the target nucleotides to obtain pure amplicon products;

in the embodiment of the invention, in addition to generating amplicon products in the PCR reaction process, a small amount of residual impurities such as enzyme, primers, dNTP, salt ions, byproducts and the like are also generated, a digestive juice is added, a program operated on a PCR instrument at 37 ℃ for 30min and 80 ℃ for 10min can remove part of amplification impurities, but the digestive juice can not completely remove double-stranded byproducts caused by amplification, purified magnetic beads are added to combine with target fragments to recover the amplicon products, and other components in the reaction are washed to remove the other components, so that a pure amplicon product is obtained, namely, the 1 st round of magnetic bead purification.

S104, taking the purified amplicon product as a template, adding a joint P5, a joint P7, a PCR mixed solution and a PCR reaction solution to perform PCR reaction to obtain a new amplicon library;

in the embodiment of the invention, the purified amplicon product is used as a template, the joint primers P5 and P7 required by high-throughput sequencing are added, P5 is a universal joint, P7 is a section of special sequence for identifying a sample, and PCR reaction is carried out by adding PCR mixed solution and PCR reaction solution. The P5 and P7 linker primers were complementary paired to the T1 and T2 sequences, respectively, flanking the amplicon product. Therefore, after the PCR reaction of this round is finished, the linker sequences P5 and P7 are respectively introduced to both sides of the amplicon product, and a new amplicon library, namely the 2 nd round linker sequence PCR reaction, is obtained.

S105, adding purified magnetic beads to combine with the target fragments to recover amplicon products, and washing to remove reaction impurities to obtain a pure amplicon library;

in the embodiment of the invention, the amplicon product is recovered by combining magnetic beads with target fragments, other components in the reaction are removed by washing, a pure amplicon library is obtained, namely, the magnetic beads in the 2 nd round are purified, and after the library is quantified and subjected to quality inspection, the amplicon library can be subjected to on-machine sequencing, namely, concentration measurement and quality inspection.

S106, carrying out high-throughput sequencing and signal-generating process analysis on the pure amplicon library to obtain sequence information of the target region, and obtaining a gene mutation result.

The implementation case is as follows: the kit of the invention specifically detects 23 gene mutations in AML through 18 clinical samples.

18 samples of patients with myeloid leukemia who are initially diagnosed, difficult to treat and relapsed (numbers A1-A18) are screened and detected, the procedures are strictly executed, a self-established birth-communication procedure is used for analysis and interpretation by professional genetic consultation unscrambler, meanwhile, the 18 samples of patients with myeloid leukemia are verified by first-generation sequencing (Sanger), the current first-generation sequencing (Sanger) is also used as a gold standard for detecting gene mutation, and the detection results of the two methods are compared as follows:

summary of the results of the multiplex PCR assay for patients with accession Nos. A1 to A9

Summary of the results of the multiplex PCR assay for patients with accession Nos. A10 to A18 (II)

Summary of the results of the patient test methods of the first generation with numbers A1-A9

Summary of results of one-generation sequencing assay methods for patients with accession numbers A10-A18 (II)

The positive rates of 23 genes detected by multiplex PCR targeting sequencing in 18 cases (accession numbers A1-A18) of acute myeloid leukemia patients who were initially diagnosed, refractory and relapsed are shown in the following table:

multiplex PCR targeted sequencing detection of 23 gene positive rate

The results of the primary sequencing tests on 23 genes of 18 cases (Nos. A1-A18) of patients with acute myeloid leukemia who were diagnosed at first time, refractory and relapsed are shown in the following table:

primary sequencing detection of 23 gene positive rate

By summarizing and comparing the results of the two detection methods, the multiple PCR targeted detection technology has higher detection rate, the first generation sequencing is limited by the sensitivity (10-15%) and methodology, the gene variation condition which is lower than 10-15% is directly judged to be negative, the result of the detected sample cannot quantify the gene mutation load rate, the multiple PCR sensitivity can reach more than 1%, and the quantitative result can be given to all detected gene mutations.

The clinical significance of some important mutant genes detected by the embodiment is shown as follows after medical interpretation of COSMIC database, ClinVar database, NCCN guideline, expert consensus in blood tumor and the like:

mutations in ASXL1 can be found in CMML, MPN, MDS, AML, and other myeloid tumors. NCCN "clinical practice guidelines for acute myeloid leukemia" in 2019 indicated that mutations in ASXL1 in AML patients suggest poor prognosis.

Mutations in BCOR are found primarily in MDS, AML. Nonsense, frameshift, splice and non-synonymous mutations of codon N1425 of the BCOR gene were indicated in NCCN "clinical practice guidelines for myelodysplastic syndrome" in 2019, with mutation rates of less than 5% in MDS patients. Is associated with poor prognosis.

Mutations in CEBPA are found mainly in AML, with mutation rates of about 7% -11% in AML patients and 13% -15% in NK-AML patients, and mainly in M1, M2 and partial M4 types of patients. NCCN "clinical practice guidelines for acute myeloid leukemia" in 2019 indicated that the concomitant presence of a cepbpa biallelic mutation alone in normal karyotype AML patients suggests a good prognosis. The single allele mutation accompanied with CEBPA has poorer prognosis than the double allele mutation accompanied with CEBPA. And CEBPA germline mutations are associated with myeloid tumor susceptibility.

Mutations in the DNMT3A gene may be found in AML, MDS and other hematological malignancies. The mutation rate in AML patients can reach 18% -22%, the mutation rate in NK-AML patients is 29% -34%, and the mutation rate is more than M5 type. The hot-spot mutation occurred with a missense variation encoding arginine 882, which combined NPM1 and FLT3 mutations in AML patients. The R882H mutation. The DNMT3A mutation is a worse prognostic factor in AML and MDS patients at risk in karyotype, or in AML patients in the ELN high-risk group. The influence of the DNMT3A mutation on prognosis is related to the mutation site, the R882 mutation AML patient has poor treatment effect, low complete remission rate and poor prognosis, and other mutations are related to the mutation site with the influence on prognosis.

Mutations in FLT3 gene are more studied in hematological disorders, occurring in a variety of myeloid tumors, accounting for approximately 1% of ALL. MDS accounts for about 5-10%, AML accounts for about 15-35%. The most common mutations are ITD (internal tandem repeat) and TKD (point mutation in the activation loop), both of which activate tyrosine kinases. The mutation rate of FLT3-ITD in AML patients is 20%, and the mutation rate in NK-AML patients can reach 28% -34%. FLT3-ITD suggests that AML has a poor prognosis, and the longer the inserted sequence, the worse the prognosis, the worse the biallelic mutation, and the higher the mutation ratio, the worse the prognosis. FLT3-TKD is found in about 7% of AML, and is mostly found in AML patients positive for APL and CBFB-MYH11, and the main mutation sites are D835, R834, I836, D839, Y842, etc.

IDH1 mutations are found mainly in AML, ALL and MPN. IDH1 has a mutation rate of about 6% -9% in AML patients and a high incidence of 8% -16% in NK-AML, R132 being the most common mutation of IDH1 in AML.

The IDH2 mutation is found primarily in karyotyped AML, as well as in MDS, MPN, and MDS/MPN catastrophe. IDH2 has a mutation rate of about 8% -12% in AML patients and a high incidence of 19% in NK-AML, occurring centrally at R140 and R172 sites.

KIT gene mutations can be found in many types of tumors, mainly in AML, mastocytosis, and NK/T cell lymphoma among hematological tumors. Its mutation rate in CBF-AML patients characterized by chromosomal t (8; 21) (q 22; q22) or inv (16) (p13.1q22) abnormalities is about 20%. KIT mutation often occurs in Exon8, 11 and 17, KIT D816V site mutation is an index of CBF-AML poor prognosis, and the disease-free survival period of a mutant patient is short. The mutation site is resistant to imatinib and effective to dasatinib.

The NPM1 mutation is mainly seen in primary AML, with an incidence of 28% -35% in AML patients and up to 48% -53% in NK-AML patients. The NPM1 Mutation is usually found in exon 12, and 6 variants are the major (named Mutation A-F). The most common of them is type A, accounting for 75% -80%. The occurrence of the NPM1 mutation alone is often an indicator of good prognosis, and the common mutation can be used as a marker for MRD monitoring.

NRAS gene mutations can be found in AML, ALL, MDS, JMML, CMML and other myeloid and lymphoid hematological tumors. Patients reporting RAS mutations in AML respond better to high doses of cytarabine with an extended overall survival and disease-free progression. NRAS germline mutations can lead to a frequently-stained predominantly inherited nononan syndrome, susceptible to myeloid tumors.

Mutations in the TET2 gene are found in a variety of myeloid tumors, and furthermore TET2 clonal hematopoiesis is present in a portion of healthy elderly individuals or patients with non-malignant hematological tumors (e.g., AA). The mutation of the TET2 gene was indicated in the clinical practice guideline for acute myeloid leukemia by NCCN in 2019 as a candidate gene for evaluating the prognosis of AML. The mutation of TET2 in normal karyotype or low-risk karyotype AML suggests poor prognosis. Demethylating agents (e.g., decitabine, azacitidine) were reported to be effective in clinical studies.

Therefore, the detection by adopting the multiple PCR targeted sequencing technology can make up the defect of first-generation sequencing, can provide economic, rapid, high-sensitivity and high-specificity gene mutation detection for clinic, and has wide clinical application value.

While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A detection kit for gene mutation in acute myeloid leukemia is characterized in that,

the kit comprises 23 genes of primer working solution, a multiplex PCR reagent, absolute ethyl alcohol, purified magnetic beads and a nucleic-FreeWater, wherein the multiplex PCR reagent comprises PCR mixed liquor, PCR reaction liquor, digestion liquor, a joint P5 and a joint P7 in a multiplex amplification system, the PCR mixed liquor comprises Tris-HCl, 10 x buffer, 4 dNTP mixtures and MgCl with the pH value of 8.3₂The PCR reaction solution comprises Taq DNA polymerase, and the digestion solution comprises shrimp alkali enzyme and exonuclease.

2. The kit for detecting a gene mutation in acute myeloid leukemia according to claim 1,

the concentration of the primer working solution of the 23 genes is 4.0-6.0 pmol/ul, the concentration of the absolute ethyl alcohol is 500ml, the concentration of the purified magnetic beads is 60ml, and the concentration of the nucleic-Free Water is 5 ml.

3. The kit for detecting a gene mutation in acute myeloid leukemia according to claim 2,

the Tris-HCl is 100mM, the Taq DNA polymerase is 3000U/ml, and the shrimp alkali enzyme and the exonuclease are 5000U/ml.

4. The kit for detecting a gene mutation in acute myeloid leukemia according to claim 3,

the joint P5 is a universal primer sequence; the adaptor P7 is a base sequence for identifying a sample.

5. The application of the detection kit for the gene mutation in the acute myeloid leukemia is characterized by comprising the following components:

6. The application of claim 5, wherein the DNA extraction kit is used for extracting DNA from a sample to be tested, the concentration of the extracted DNA is greater than or equal to 10ng/ul, and the method specifically comprises the following steps:

7. The use of claim 6, wherein purifying the amplicon product of the plurality of target nucleotides to obtain a purified amplicon product comprises: