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

A technology of primer sets and kits, applied in the field of primer sets for detection of AML-related gene mutations, can solve the problems of low Sanger sequencing sensitivity, undetectable samples with low mutation frequencies, and difficulty in detecting low-frequency mutations. Efficient, time-saving, and error-reducing effects

Active Publication Date: 2020-04-10
CARRIER GENE TECH SUZHOU CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Sanger sequencing has low sensitivity and cannot detect samples with low mutation frequency
[0005] Among the current methods for detecting AML gene mutations at home and abroad, fluorescence in situ hybridization (FISH) can only be used for qualitative detection, and the operation is complicated; while polymerase chain reaction (Polymerase Chain Reaction, PCR) technology combined with Sanger sequencing technology However, it has the limitation of low sensitivity, and it is difficult to detect low-frequency mutations; although NGS technology can detect qualitatively and quantitatively, and has high sensitivity, it has long detection time, high cost, and high data requirements for low-frequency mutations. limitations

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

Examples

Experimental program
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Embodiment 1

[0074] This embodiment provides a primer set for detecting AML-related gene mutations, which includes a pair of primers for the FLT3 gene, a pair of primers for the DNMT3A gene, a pair of primers for the IDH1 gene, a pair of first primers for the IDH2 gene, a pair of second primers for the IDH2 gene, and KIT A pair of gene primers and a pair of NPM1 gene primers; the nucleotide sequence of the FLT3 gene primer pair is shown in the sequence listing SEQ ID NO: 1-2; the nucleotide sequence of the DNMT3A gene primer pair is shown in the sequence listing SEQ ID NO : shown in 4-5; the nucleotide sequence of the IDH1 gene primer pair is shown in the sequence table SEQ ID NO: 7-8; the nucleotide sequence of the first primer pair of the IDH2 gene is shown in the sequence table SEQ ID NO : shown in 10~11; The nucleotide sequence of the second primer pair of IDH2 gene is shown in the sequence listing SEQ ID NO:13~14; ​​The nucleotide sequence of the KIT gene primer pair is shown in the se...

Embodiment 2

[0081]This embodiment provides a test kit for detecting AML-related gene mutations, which includes PCR amplification reaction reagents, positive quality control products, negative quality control products and standard curve equations, wherein the test kit also includes the above-mentioned A primer set and a primer probe set corresponding to the primer set. The corresponding gene primer pairs between the primer sets exist in the form of a mixed solution, and the probes of the primer-probe set can exist in the form of a mixed solution with the primer pairs of the corresponding genes. The molar concentration of each primer in the primer set is 2-100 μmol / L, and the molar concentration of each probe in the probe set is 10-1000 μmol / L. The probe has an overlapping sequence of 5-13 bp with the upstream primer in the primer pair of the corresponding gene, and 4 mismatched bases are designed at the 3' end of the probe or C3 Spacer modification is performed. When the probe and the wil...

Embodiment 3

[0087] This embodiment provides a method for detecting AML-related gene mutations using the kit provided in the above-mentioned embodiment 2. In each round of detection reaction, the samples should be analyzed simultaneously with the positive quality control and the negative quality control. It includes the following steps:

[0088] (1) Obtain the DNA of the sample to be tested as a template; specifically, the DNA OD260 / OD280 value should be between 1.8 and 2.0, dilute the DNA concentration to 10 ng / μL, and immediately test the template or store it at -20°C spare.

[0089] (2) Mix the above-mentioned primer-probe group, the above-mentioned PCR amplification reaction reagent and template together for PCR amplification reaction to obtain the first CT value; specifically, first take 6 μL of the seven sets of primer probes provided in Example 2 above The mixed solution, seven groups of 25 μL PCR amplification reaction reagents and seven groups of 9 μL nuclease-free water were pre...

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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 an FLT3 gene primer pair, a DNMT3A gene primer pair, an IDH1 gene primer pair, an IDH2 gene first primer pair, an IDH2 gene second primer pair, a KIT gene primer pair and an NPM1gene primer pair; the kit provided by the invention comprises the primer group and one or more of 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 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 quantitatively analyzing the AML related gene mutation frequency.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a primer set, a kit and a method for detecting AML-related gene mutations. Background technique [0002] Acute myeloid leukemia (AML) is a genetically heterogeneous disease whose clinical course can be predicted by recurrent cytogenetic abnormalities and / or gene mutations. Cytogenetic detection of specific karyotype abnormalities is often considered a marker to guide treatment and judge prognosis. However, there are also some patients with undetectable karyotype abnormalities, and these patients have large variations in treatment response or prognosis evaluation. With the rapid development of molecular biology techniques, multiple gene mutations related to AML progression or AML prognosis have been identified, which fully proves that gene mutations are an important source of AML pathogenesis. [0003] In AML, IDH mutations are associated with undifferentiated acute myelo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2545/114C12Q2531/113C12Q2535/101
Inventor 徐雪成昱璇宋萍汪进平陈苗苗王方金杨玉霞罗俊峰
Owner CARRIER GENE TECH SUZHOU CO LTD
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