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A primer, probe and kit for detecting flt3 gene mutation

A technology for detecting kits and probes, which is applied in biochemical equipment and methods, measurement/inspection of microorganisms, DNA/RNA fragments, etc. It can solve the problems of reducing the accuracy of kits, taking a long time, and detecting false positives, and achieves Broad application prospects and industrialization prospects, the detection process is simple and efficient, and the effect of simplifying the detection steps

Active Publication Date: 2021-12-17
广东永诺医疗科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these detection methods have some disadvantages
For example, the restriction endonuclease method cannot be 100% effective in recognizing wild-type DNA for cutting, which is likely to cause false positive results and reduce the accuracy of the kit, especially when the mutation rate in the sample is low. The accuracy and sensitivity of judging whether there is a mutation in the sample by cleavage with endonucleases are very low; the detection throughput of traditional Sanger sequencing is low, and the detection sensitivity is not high: 1%~10%
Although the detection throughput of NGS is large, the detection sensitivity is greatly improved compared with capillary electrophoresis, but the cost of NGS method is expensive, the data analysis is complicated and time-consuming, and the sensitivity is only about 1%.

Method used

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  • A primer, probe and kit for detecting flt3 gene mutation
  • A primer, probe and kit for detecting flt3 gene mutation
  • A primer, probe and kit for detecting flt3 gene mutation

Examples

Experimental program
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Effect test

Embodiment 1

[0031] A kind of primer, probe and kit for detecting FLT3 gene mutation of the embodiment of the present invention, it comprises:

[0032] (1) Reaction master mix for droplet digital PCR: including buffer, dNTPs, taq enzyme, UDG enzyme;

[0033](2) FLT3-TKD detection mixture: including a pair of primers and two probe combinations for detecting the two sites of FLT3 D835 and I836; the primers include primers for targeted detection of FLT3 gene D835 site mutations and targeted detection of FLT3 The primer of gene I836 site mutation, its sequence is as shown in SEQ ID NO:1 and SEQ ID NO:2; Wherein the probe includes specificity probe and internal reference probe, and its sequence is as SEQ ID NO:3 and SEQ ID NO:4 shown;

[0034] (3) FLT3 D835 Weak Positive Control: It is prepared in proportion to the recombinant plasmid containing the FLT3 D835 mutant gene fragment and the FLT3 wild-type plasmid diluted with 1*TE; the nucleotide sequence of the FLT3 wild-type plasmid is shown in...

Embodiment 2

[0039] A detection method of a kit for detecting FLT3 gene mutation according to Embodiment 1 of the present invention, comprising the following steps:

[0040] (1) Sample acquisition: The applicable sample types are whole blood or bone marrow fluid.

[0041] (2) Sample processing and nucleic acid extraction: purchase a commercial nucleic acid extraction kit, perform DNA extraction on clinical samples according to the instructions of the nucleic acid extraction kit, and use Nano-Drop to measure the nucleic acid concentration of the extracted DNA solution;

[0042] (3) Reagent preparation:

[0043] a. Set positive quality control and blank control quality control for detection reaction.

[0044] b. Preparation process

[0045] 1) Thaw all components to room temperature, shake and mix evenly after each component is fully dissolved, and centrifuge briefly;

[0046] 2) Determine the number of reactions N, N=number of samples to be tested (n)+number of quality controls (3)+1. T...

Embodiment 3

[0069] Example 3 Optimization of the components of the reaction premix

[0070] The embodiment of the present invention tested and screened the components of the reaction premix for droplet digital PCR in the kit for detecting FLT3 gene mutation in embodiment 1 of the present invention. Two different droplet digital PCR reaction master mixes mix1 and mix2 were prepared for testing. The specific steps are as follows:

[0071] (1) Use plasmids to prepare samples of different mutant types:

[0072] a. Take out the diluted FLT3 D835 plasmid, FLT3 I836del plasmid and FLT3 wild-type plasmid solutions from the -20°C refrigerator, dissolve them, shake and mix well;

[0073] b. Dilute the mutant FLT3 D835 plasmid solution 10 times with the FLT3 wild-type plasmid solution to prepare: take 30ul mutant FLT3 D835 plasmid and add 270ul wild-type plasmid to mix, and name it as FLT3 D / WT plasmid 10%;

[0074] c. Dilute the mutant FLT3 I836del plasmid solution 10 times with the FLT3 wild-typ...

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Abstract

The invention discloses a primer, a probe and a kit for detecting FLT3 gene mutation, belonging to the field of gene detection. The primers and probes for detecting FLT3 gene mutations of the present invention include primers and probes for targeted detection of FLT3 gene D835 site mutations and FLT3 gene I836 site mutations. The primer and kit of the present invention rely on digital PCR detection technology, and compared with other current technologies for detecting FLT3 mutations, it simplifies the detection steps and has the advantages of simple and efficient detection process, absolute quantification, and high sensitivity. The invention provides strong technical support for auxiliary diagnosis and clinical treatment effect monitoring of AML patients carrying FLT3 gene D835 or I836del mutation, and has broad application prospects and industrialization prospects.

Description

technical field [0001] The invention belongs to the field of gene detection, and in particular relates to a primer, a probe and a kit for detecting FLT3 gene mutation. Background technique [0002] The FLT3 gene is located on chromosome 13q12.2 and has 24 exons. The encoded protein belongs to a member of the type III receptor tyrosine kinase family. The full name is FMS-like tyrosine kinase 3 (FMS-like tyrosine kinase 3). Plays an important role in the survival, proliferation and differentiation of hematopoietic cells. The FLT3 protein consists of five structures including an immunoglobulin-like extracellular domain located in the extracellular region, a transmembrane domain, a juxtamembrane domain (JMD), a tyrosine kinase domain interruption (TKD) and a small C-terminal domain. domain composition. According to the 2015 China Cancer Statistics Report, the incidence of leukemia in China is 75,300 per year, and the death toll is 53,400 per year, among which adult acute myelo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6851C12N15/11
CPCC12Q1/6886C12Q1/6851C12Q2600/156C12Q2600/118C12Q2600/166C12Q2531/113C12Q2545/101C12Q2563/159C12Q2561/101
Inventor 罗景燕李静芳许少飞赖炳权
Owner 广东永诺医疗科技有限公司