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Primer composition for detecting AML prognosis related gene mutation, and kit and application thereof

A primer composition and kit technology, applied in recombinant DNA technology, biochemical equipment and methods, and microbial determination/inspection, etc., can solve problems such as small flux, complex process, easy recurrence, etc., and achieve high accuracy of results. , the detection process is simple, the effect of wide application prospects

Pending Publication Date: 2020-08-21
QIAGEN SUZHOU TRANSLATIONAL MEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The 5-year long-term survival rate of AML patients is less than 50%. Even patients with good prognosis are prone to relapse, and the prognosis after relapse is extremely poor.
[0007] At present, the technologies for simultaneous detection of FLT3-ITD, FLT3-TKD, NPM1 and CEBPA mainly include NGS technology, PCR+capillary electrophoresis technology, q-PCR technology, pyrosequencing technology and agarose gel electrophoresis technology, but there are more or less Some disadvantages: NGS technology has a long detection cycle and high cost; PCR+capillary electrophoresis technology mainly uses Sanger sequencing technology and fragment analysis technology. Carry out PCR amplification for each gene separately, and use it on the machine alone or in combination. At present, mature products generally only include 1-2 genes, and the coverage is not comprehensive; q-PCR technology has a small throughput and is not suitable for multi-gene detection; Pyrosequencing technology has complicated operation process and low throughput; agarose gel electrophoresis technology cannot clearly distinguish short fragment insertions, and false negative results are prone to occur
[0008] At present, the detection of FLT3-ITD, FLT3-TKD, NPM1 and CEBPA at home and abroad mainly uses DNA and RNA as the operating object, and FLT3-ITD uses RNA as the operating object, which will result in an incomplete detection range, because ITD mutations include intron insertions
In addition, RNA is easily contaminated and degraded, and the efficiency of reverse transcription is low, which affects the detection sensitivity

Method used

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  • Primer composition for detecting AML prognosis related gene mutation, and kit and application thereof
  • Primer composition for detecting AML prognosis related gene mutation, and kit and application thereof
  • Primer composition for detecting AML prognosis related gene mutation, and kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1 Primer Design

[0076] FLT3 gene mutations mainly include internal tandem duplication mutations in the transmembrane region (FLT3-ITD) and point mutations involving the activation loop (FLT3-TKD), where the ITD region includes exon 14, intron 14 and exon 15 Exon mutation, the TKD region includes D835 and I836 mutations, and can be recognized by the restriction endonuclease EcoR V. This embodiment is designed for FLT3-ITD and FLT3-TKD such as SEQ ID NO: 1-2 and SEQ ID NO: 3 The primer pair shown in ~4;

[0077] The vast majority of NPM1 mutations include the insertion of 4 bases in exon 12. The insertion types mainly include A, B, C, D, E, and F, and the positions are relatively fixed. Any type of insertion can cause changes in the fragment length. Embodiment Design primer pairs as shown in SEQ ID NO: 5-6 for above-mentioned 6 kinds of mutant regions;

[0078] CEBPA gene mutations mainly occur near the start codon, the middle of the gene, and the stop codon. ...

Embodiment 2

[0104] Embodiment 2 kit composition

[0105] In this example, based on the primer composition designed in Example 1, a kit for AML prognosis-related gene mutation was constructed, as shown in Table 1.

[0106] Table 1

[0107]

[0108]

Embodiment 3A

[0109] Embodiment 3 AML mutation site detection system

[0110] The flow chart of AML mutation site detection is as follows: figure 1 As shown, the extracted DNA samples were amplified by multiplex PCR using two PCR systems respectively, the amplified products of PCR system 1 were digested, and the digested products and undigested products were tested on the machine. The results were analyzed for data.

[0111] Two systems were used to detect all mutation sites of FLT3, NPM1 and CEBPA. The total amount of DNA template was 500ng, and the DNA was eluted with water during extraction to avoid the inhibition of enzyme activity by EDTA in TE buffer when the DNA concentration was too low;

[0112] PCR system 1 mixed FLT3-ITD, FLT3-TKD, NPM1 and CEBPA-2 primers for PCR, PCR system 2 mixed CEBPA-1 and CEBPA-3 primers for PCR, PCR systems 1 and 2 were prepared according to Table 2, PCR System 1 and 2 run the program according to Table 3;

[0113] Table 2

[0114] component...

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Abstract

The invention provides a primer composition for detecting AML prognosis related gene mutation, and a kit and an application thereof. The primer composition comprises six primer pairs for detecting FLT3-ITD mutation, FLT3-TKD mutation, NPM1 mutation and CEBPA mutation, all the primer pairs are matched with one another, comprehensive detection of AML prognosis related mutant genes is achieved, and the primer composition is easy and convenient to operate, high in sensitivity and good in accuracy.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and relates to a primer composition, a kit and an application thereof for detecting mutations of genes related to AML prognosis. Background technique [0002] Acute myeloid leukemia (AML) is a group of heterogeneous hematopoietic malignancies characterized by uncontrolled proliferation of hematopoietic stem cells, blockage of differentiation, and inhibition of normal hematopoietic function. entity. The 5-year long-term survival rate of AML patients is less than 50%. Even patients with good prognosis are prone to relapse, and the prognosis after relapse is extremely poor. With the deepening of research, it is found that gene mutation is one of the key factors affecting the prognosis of AML. [0003] The WHO 2008 guidelines for the classification of myeloid neoplasms and acute leukemia recommend NPM1, FLT3, and CEBPA gene screening for all patients with normal karyotype acute myeloid leuke...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/16C12Q2600/118C12Q2600/156C12Q2531/113C12Q2563/107C12Q2537/143C12Q2565/125C12Q2521/301
Inventor 辛秋红王洁吴建成张亚飞
Owner QIAGEN SUZHOU TRANSLATIONAL MEDICINE CO LTD
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