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Joint detection method and diagnostic kit of NPM1 (Nucleophosmin 1) gene mutation

A diagnostic kit and gene technology, applied in the direction of fluorescence/phosphorescence, material excitation analysis, etc., can solve the problems of cumbersome operation, poor repeatability, and insufficient sensitivity, and achieve good stability, high specificity, and rapid detection.

Active Publication Date: 2013-01-23
MEDI GENETECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Immunological methods have high false positive and false negative rates, and cannot achieve early diagnosis
Among the molecular biology methods widely used at home and abroad to detect leukemia gene mutations, fluorescence in situ hybridization (FISH) can only perform qualitative detection, and the operation is complicated; fluorescent quantitative PCR has limitations in detection throughput, so none of them can Really meet the needs of clinical diagnostic testing
The traditional solid-phase biochip (Biochip) technology has outstanding weaknesses such as poor repeatability, insufficient sensitivity, and cumbersome operations.

Method used

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  • Joint detection method and diagnostic kit of NPM1 (Nucleophosmin 1) gene mutation
  • Joint detection method and diagnostic kit of NPM1 (Nucleophosmin 1) gene mutation
  • Joint detection method and diagnostic kit of NPM1 (Nucleophosmin 1) gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1: A liquid-phase chip detection method for NPM1 gene mutation

[0067] The specific detection method includes the following steps:

[0068] 1. Preparation of microsphere mixture for detection of NPM1-mutA mutant gene

[0069] 1. Synthesize oligonucleotide probes according to the following sequence:

[0070] NPM1-mutA: 5'-AminolinkerC12 CCTCCACTGCCAGACAGAGATC-3', as shown in SEQ ID NO.1;

[0071] Abelson gene: 5'-AminolinkerC12 CTGAAGGGCTTCTTCCAGAT-3', as shown in SEQ ID NO.12;

[0072] 2. Coupling the above oligonucleotide probes containing amino modifications with two kinds of carboxyl microspheres numbered 11 and 21 respectively

[0073] 2.1 Take out a small portion of fresh dry powdered EDC stored at -20°C and equilibrate to room temperature;

[0074] 2.2 with dH 2 O dissolves the oligonucleotide probes of NPM1-mutA and Abelson respectively, the concentration is 1mM (1nmol / μl);

[0075] 2.3 Vortex the storage suspensions of the two carboxyl microsphere...

Embodiment 2

[0172] Example 2: Liquid-phase chip joint parallel detection method for two kinds of NPM1 gene mutations

[0173] The specific detection method includes the following steps:

[0174] 1. Preparation of microsphere mixture for detecting NPM1-mutA and NPM1-mutB mutant genes

[0175] 1. Synthesize oligonucleotide probes according to the following sequence:

[0176]NPM1-mutA: 5'-AminolinkerC12 CCTCCACTGCCAGACAGAGATC-3', as shown in SEQ ID NO.1;

[0177] NPM1-mutB: 5'-AminolinkerC12 CTCCACTGCCATGCAGAGATC-3', as shown in SEQ ID NO.2;

[0178] Abelson gene: 5'-AminolinkerC12 CTGAAGGGCTTCTTCCAGAT-3', as shown in SEQ ID NO.12;

[0179] 2. Coupling the above oligonucleotide probes containing amino modifications to the three kinds of carboxyl microspheres numbered 11, 15, and 21 respectively

[0180] 2.1 Take out a small portion of fresh dry powdered EDC stored at -20°C and equilibrate to room temperature;

[0181] 2.2 with dH 2 O dissolves the oligonucleotide probes of NPM1-mutA, N...

Embodiment 3

[0282] Example 3: Liquid-phase chip joint parallel detection method for three kinds of NPM1 gene mutations

[0283] The specific detection method includes the following steps:

[0284] 1. Preparation of microsphere mixture for detecting NPM1-mutA, NPM1-mutB, NPM1-mutD mutant genes

[0285] 1. Synthesize oligonucleotide probes according to the following sequence:

[0286] NPM1-mutA: 5'-AminolinkerC12 CCTCCACTGCCAGACAGAGATC-3', as shown in SEQ ID NO.1;

[0287] NPM1-mutB: 5'-AminolinkerC12 CTCCACTGCCATGCAGAGATC-3', as shown in SEQ ID NO.2;

[0288] NPM1-mutD: 5'-AminolinkerC12 TCCACTGCCAGGCAGAGATC-3', as shown in SEQ ID NO.3;

[0289] Abelson gene: 5'-AminolinkerC12 CTGAAGGGCTTCTTCCAGAT-3', as shown in SEQ ID NO.12;

[0290] 2. Coupling the above oligonucleotide probes containing amino modifications to the four kinds of carboxyl microspheres numbered 11, 15, 17, and 21 respectively

[0291] 2.1 Take out a small portion of fresh dry powdered EDC stored at -20°C and equilibra...

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Abstract

The invention discloses a joint detection method and a diagnostic kit of nucleophosmin (NPM1) gene mutation. Primers and probes are designed for NPM1 mutant genes including NMP1-mutA, NMP1-mutB and NMP1-mutD, a probe microsphere mixture formed by the convalent binding of the probes and microspheres is hybridized with a reverse transcription PCR (Polymerase Chain Reaction) product, and after streptavidin and rhodophyll protein are added, the fluorescence signals of different microspheres can be detected, and thus, whether a sample to be detected comprises NPM1 gene mutation and the expression conditions of the mutant genes can be determined. The method and the kit have the advantages of high sensitivity, high throughput, rapid detection, accuracy and the like, and can qualitatively and quantitatively detect NPM1 gene mutation; an important reference basis is provided for determining the early diagnosis, the recurrence and the treatment scheme of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) and judging prognosis.

Description

technical field [0001] The invention relates to the technical field of in vitro diagnosis and detection, in particular to a liquid-phase chip combined detection method for NPM1 gene mutation and a diagnostic kit thereof. Background technique [0002] The nucleophosmin (NPM) gene is located on human chromosome 5q35, contains 12 exons, and encodes a NPM protein consisting of 294 amino acids. NPM1 is involved in ribosome synthesis and centrosome duplication to regulate cell proliferation and cycle progression. NPM1 mutations only involve exon 12, and there are currently more than 40 different mutation subtypes, most of which contain the insertion of 4 bases in exon 12. The most common one is NPM1-mutA, which accounts for 75%-80% of all mutations. It is a tandem repeat sequence formed by inserting a TCTG4 nucleotide at position 956-959 of the wild-type NPM1 nucleotide sequence. NPM1-mutB and NPM1-mutD accounted for 10% and 5% respectively, and the insertions were CATG and CCTG...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 邵棠陈鸣
Owner MEDI GENETECH