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Fluorescent PCR (Polymerase Chain Reaction) detection kit for IDH1/IDH2 (isocitrate dehydrogenase 1/isocitrate dehydrogenase 2) gene mutation and application thereof

A technology of IDH2 and IDH1, which is applied in the direction of DNA/RNA fragments, microbial measurement/inspection, recombinant DNA technology, etc., can solve the problems of time-consuming, cumbersome steps, complicated procedures, etc., and achieve simple and fast operation, high sensitivity, and improved Effect of Detection Sensitivity

Inactive Publication Date: 2013-12-11
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The direct sequencing method to detect gene mutations has the disadvantages of cumbersome steps, complex procedures, time-consuming and labor-intensive

Method used

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  • Fluorescent PCR (Polymerase Chain Reaction) detection kit for IDH1/IDH2 (isocitrate dehydrogenase 1/isocitrate dehydrogenase 2) gene mutation and application thereof
  • Fluorescent PCR (Polymerase Chain Reaction) detection kit for IDH1/IDH2 (isocitrate dehydrogenase 1/isocitrate dehydrogenase 2) gene mutation and application thereof
  • Fluorescent PCR (Polymerase Chain Reaction) detection kit for IDH1/IDH2 (isocitrate dehydrogenase 1/isocitrate dehydrogenase 2) gene mutation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Design and verification of primers and probes

[0049] 1 Experimental method

[0050] 1.1 Collection of template DNA

[0051] The paraffin-embedded tissue sections or frozen tissue blocks of 50 patients with glioblastoma diagnosed clinically and pathologically were collected, and genomic DNA was extracted from the tissues for the following experimental applications. Fluorescent PCR was used to detect 7 common base substitution mutations at codon 132 of IDH1 gene and codon 172 of IDH2 gene.

[0052] 1.2 Primer design

[0053] Design and screen one specific ARMS primer and one reference primer that can specifically detect the above seven base substitution mutations, design and screen a general downstream primer, design and screen a general TaqMan probe, design and screen a nucleic acid amplification blocking primer One, the sequences of each probe and primer are as follows:

[0054] The sequence of IDH1 nucleic acid amplification blocking primer is shown in ...

Embodiment 2

[0083] Embodiment 2 Kit 1

[0084] The kit contains the following reagents and materials:

[0085] Primer solution: (1) IDH1 nucleic acid amplification retardation primer, sequence SEQ ID NO.1, 5 μmol / L; (2) ARMS primer, sequence is any one or several in SEQ ID NO.6-9, 5 μmol / L L; (3) Universal TaqMan probe for IDH1 mutation detection, sequence SEQ ID NO.3, 5 μmol / L; (4) reference primer, sequence SEQ ID NO.5, 5 μmol / L.

Embodiment 3

[0086] Example 3 Kit 2

[0087] The kit contains the following reagents and materials:

[0088] Primer solution: (1) IDH2 nucleic acid amplification retardation primer, sequence SEQ ID NO.2, 5 μmol / L; (2) ARMS primer, sequence is any one or several in SEQ ID NO.10-12, 5 μmol / L L; (3) Universal TaqMan probe for IDH2 mutation detection, sequence SEQ ID NO.4, 5 μmol / L.

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Abstract

The invention relates to a fluorescent PCR detection kit for the IDH1 / IDH2 (isocitrate dehydrogenase 1 / isocitrate dehydrogenase 2) gene mutation and application thereof, and particularly provides a nucleotide sequence used for detecting the IDH1 / IDH2 gene mutation, a kit with the nucleotide sequence and the application of the kit to detection of the IDH1 / IDH2 gene mutation. The nucleotide sequence comprises a specific ARMS (Amplification Refractory Mutation System) primer, a general mutation detection TaqMan probe and a nucleic acid amplification retardation primer. The kit can be used for quickly detecting the IDH1 / IDH2 gene mutation with high throughput and at a low cost, is high in sensitivity, good in specificity, low in pollution and quick and safe to operate, can be suitable for high-sensitivity detection of trace mutation in general clinic samples such as fresh frozen tissues and paraffin tissues, especially non-traumatic serums or plasma samples in addition to pathological tissues.

Description

technical field [0001] The invention relates to the technical field of molecular biology diagnosis, in particular to a fluorescent PCR detection kit for IDH1 / IDH2 gene mutation and its application. Background technique [0002] The IDH (isocitrate dehydrogenase) family in mammalian cells includes 3 members: IDH1, IDH2 and IDH3. IDH1 exists in the cytoplasm and peroxisomes, and IDH2 and IDH3 exist in the mitochondria. All three enzymes catalyze the oxidative decarboxylation of isocitrate (ICT) to produce CO 2 and α-ketoglutarate (α-KG). These reactions require divalent ions (Mg 2+ or Mn 2+ ) is involved, and requires the cofactor NADP + (IDH1 and IDH2) or NAD + (IDH3) acts as an electron acceptor to generate NADPH or NADH, respectively. IDH1 and IDH2 are structurally similar isomerases, whereas IDH3 is different from IDH1 and IDH2 in that its enzymatic structure is a heterotetraploid protein including two IDH3A subunits, one IDH3B subunit and one IDH3G subunit. [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 董艳韩晞盛平
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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