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Primer, probe and kit for detecting FLT3 gene mutation

A kit and probe technology, applied in the field of gene detection, can solve the problems of reducing the accuracy of the kit, time-consuming, false positive detection, etc., and achieve broad application prospects and industrialization prospects, the detection process is simple and efficient, and the detection process is simplified. effect of steps

Active Publication Date: 2021-10-15
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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  • Primer, probe and kit for detecting FLT3 gene mutation
  • Primer, probe and kit for detecting FLT3 gene mutation
  • Primer, probe and kit for detecting FLT3 gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] A primer, probe, and kit for detecting FLT3 gene mutation, including:

[0032] (1) Microphone-based digital PCR recombine with reaction: including buffer, DNTPS, TAQ enzyme, UDG enzyme;

[0033](2) FLT3-TKD detection mixture: including a pair of primers and two probes combinations of two sites of FLT3 D835 and I836; in which primers include targeted primers and targeted detection of FLT3 gene D835 site mutations. The primer of the gene I836 site mutation is shown in SEQ ID NO: 1 and SEQ ID NO: 2; wherein the probe comprises a specific probe and an inner probe whose sequence such as SEQ ID NO: 3 and SEQ ID NO: 4 shows;

[0034] (3) FLT3 D835 Weak positive control: a recombinant plasmid containing FLT3 D835 mutation gene fragment, 1 * TE dilution is prepared, and the nucleotide sequence of FLT3 wild plasmid, such as sequence list SEQ ID NO : 5 shows the nucleotide sequence of FLT3 D835 mutant recombinant plasmid, such as sequence list SEQ ID NO: 6;

[0035] (4) FLT3 I836DEL w...

Embodiment 2

[0039] The detection method for detecting a kit for detecting a FLT3 gene mutation, comprising the steps of:

[0040] (1) Sample acquisition: Applicable sample type is acute or bone marrow liquid.

[0041] (2) Sample processing and nucleic acid extraction: Purchase commercial nucleic acid extraction kits, extracts the clinical sample by the nucleic acid extraction kit specification, and the extracted DNA solution was measured by NaNO-DROP;

[0042] (3) Reagent formulation:

[0043] a. Detection reaction sets the yang property control and blank control.

[0044] b. Formulation process

[0045] 1) Decompose all groups to room temperature, and each component is fully dissolved and mixed and mixed, and the centrifugation is short;

[0046] 2) Determine the number of reaction N, N = to be inspected (N) + number of quality control (3) +1. The amount of each reagent added to the reaction mixture was calculated, and the calculation was calculated as shown in Table 1.

[0047] Table 1

[0...

Embodiment 3

[0069] Example 3 Optimization of Reaction Premix Components

[0070] The embodiment of the present invention is tested for testing the premixing ingredient of the reaction premixing ingredient for detecting the kit of the Example 1 of the present invention. Two different microphena-based PCRs were prepared with the reaction premixing solution Mix1 and Mix2, and the specific steps are as follows:

[0071] (1) Preparation of different mutant samples with plasmid:

[0072] a. Remove the FLT3 D835 plasmid, FLT3 I836DEL plasmid and FLT3 wild plasmid solution, which is diluted, and after dissolving, and mixed, lysed.

[0073] b. Dilute the mutant FLT3 D835 plasmid solution to dilute 10 times the formation of FLT3 wild plasmid solution: Take 30 ul mutant FLT3 D835 plasmid to add 270 ul wild-type plasmid mix, named FLT3 D / WT plasmid 10%;

[0074] c. Dilute the mutant FLT3 I836DEL plasmid solution 10 times with FLT3 wild plasmid solution: Take 30 ul mutant FLT3 I836DEL plasmid to mix the...

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Abstract

The invention discloses a primer, a probe and a kit for detecting FLT3 gene mutation, and belongs to the field of gene detection. The primer and the probe for detecting FLT3 gene mutation comprise a primer and a probe for specifically detecting FLT3 gene D835 site mutation and FLT3 gene I836 site mutation. The primer and the kit provided by the invention depend on a digital PCR detection technology, compared with other FLT3 mutation detection technologies at present, the detection steps are simplified, and the primer and the kit have the advantages of simple and efficient detection process, absolute quantification, high sensitivity and the like. Powerful technical support is provided for auxiliary diagnosis and clinical treatment effect monitoring of AML patients carrying FLT3 gene D835 or I836del mutation, and wide application prospects and industrialization prospects are achieved.

Description

Technical field [0001] The present invention belongs to the field of gene detection, and more particularly to primers, probes, and kits for detecting FLT3 gene mutation. Background technique [0002] The FLT3 gene is located in chromosome 13q12.2, with 24 exons, and the encoded protein belongs to one of the members of the III receptor tyrosine kinase family, and it is full name of FMS sample type tyrosine kinase 3 (FMS-LIKE TYROSINE KINase 3), It plays an important role in the survival, proliferation and differentiation of hematopoietic cells. FLT3 proteins are 5 structures such as immunoglobulin-like extracellular domains, transmembrane domains, proximal domains (JMDs), tyrosine kinase domains (TKD), and small C-terminal domains. Domain consists. According to 2015 China's tumor statistical report, the number of Chinese leukemia has a number of 75,300 / year, and the number of deaths is 53,400 / year. Among them, the adult acute myeloside leukemia (AML) accounts for 65% of adult ...

Claims

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Application Information

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Patent Type & Authority Applications(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