Primer pair, probe set and kit for detecting mitochondrial obesity gene mutation

An obesity gene and mitochondrial technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/testing, etc., can solve problems such as incomplete coverage, high price, and racial differences, and speed up clinical diagnosis Efficiency, high clinical scalability, and short detection time period

Pending Publication Date: 2021-08-31
北京华诺奥美基因生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, relevant research at home and abroad is based on the distribution of clinical phenotypes of their respective cohorts of patients, limited scope, racial differences, and the coverage of each study is not completely the same
The detection method of tRNA Leu(UUR)3243A>G mutation has the traditional PCR digestion method, which is complicated to operate and has cumbersome steps
The method of high-throughput sequencing can also be used, but it is expensive; while the method of fluorescent quantitative PCR has the characteristics of simple operation, convenience and quickness
[0005] Related technologies disclose the use of fluorescent quantitative PCR method to qu

Method used

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  • Primer pair, probe set and kit for detecting mitochondrial obesity gene mutation
  • Primer pair, probe set and kit for detecting mitochondrial obesity gene mutation
  • Primer pair, probe set and kit for detecting mitochondrial obesity gene mutation

Examples

Experimental program
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Effect test

Embodiment

[0032] 1. Nucleic acid detection kit for mitochondrial obesity gene tRNA Leu(UUR)3243A>G mutation site (fluorescence quantitative PCR).

[0033] The present invention has established a kind of utilizing fluorescent quantitative PCR technology to be simple, fast, accurate, high-throughput detection human mitochondrial tRNA Leu (UUR) 3243A>G mutation site primer pair, probe group and kit, thereby for tRNA Leu (UUR) )3243A>G mutation detection provides a reference, and the kit is stored at -20°C.

[0034] Kit specifications: 50 servings / box, see Table 1 for specific components.

[0035] Table 1. Kit components

[0036]

[0037] Detection solution: including the first primer pair for tRNA Leu (UUR) 3243 site, the first probe set and RNase FreeddH 2 O.

[0038] PCR amplification reaction solution: purchased from Nanjing Novizan Biotechnology Co., Ltd., product number: Q113-02 / 03.

[0039] Positive control substance: a plasmid containing the mitochondrial tRNA Leu(UUR)3243G s...

experiment example

[0076] Experimental example: detection of 50 clinical samples.

[0077] 1. Sample processing

[0078] The blood samples of 10 diabetics and 40 healthy subjects were obtained, and the samples were collected from a hospital.

[0079] Key points for sample acquisition: Use purple cranial tubes (blood collection tubes containing ethylenediaminetetraacetic acid and salt) to collect venous blood, and store it at 4°C for later use.

[0080] Take 200 μL of each sample, and perform nucleic acid extraction and DNA extraction according to the Bio-Nucleic Acid Extraction Kit (product number: IVD4173) for later use.

[0081] 2. PCR amplification

[0082] Configure the PCR amplification reaction solution as shown in the table below (20 μL for each reaction)

[0083] components 1 reaction volume 60 Detection solution 7.5μL 450μL Amplification reaction solution 12.5μL 750μL total capacity 20 μL 1200μL

[0084] Aliquot the prepared PCR amplificat...

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Abstract

The invention belongs to the technical field of biological detection, and discloses a primer pair, a probe set and a kit for detecting mitochondrial obesity gene mutation. The kit takes human DNA as a formwork, fluorescence quantitative PCR amplification is carried out to obtain an amplification curve, and a mitochondrial tRNA Leu (UUR) 3243A more than G mutation site can be quickly, accurately and sensitively detected. The kit is further provided with a wild type site probe, and the relative ratio of a mutant type to a wild type is obtained through Ct values of the mutant type and the wild type probe. The blank of clinically detecting the tRNA Leu (UUR) 3243A more than G mutation site is filled, and timely auxiliary data is provided for subsequent treatment of related diseases. The kit is good in repeatability and high in precision. The kit is short in detection period, detection can be completed within 45 minutes at the soonest, the detection time is saved, the efficiency is improved, and the kit is an efficient auxiliary detection means. In the application of non-diagnosis purposes, detection can be completed only by mixing the kit and a collected sample through a fluorescence quantitative PCR instrument, the operation requirement is simple, and the clinical popularization performance is high.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a primer pair, a probe set and a kit for detecting mitochondrial obesity gene mutations. Background technique [0002] Mitochondrial disease refers to the abnormality of mitochondrial oxidative respiratory chain-related proteins due to mutations or deletions of mitochondrial DNA and nuclear DNA, which in turn leads to multiple organ dysfunction syndrome that hinders ATP synthesis. The types of mitochondrial gene mutations include base mutations, insertion and deletion mutations, and mtDNA copy number mutations. tRNA Leu(UUR)3243A>G is the most common mitochondrial gene mutation, which is usually maternally inherited, but sporadic mutations can occasionally lead to A series of clinical symptoms ranging from mild to fatal, one of which is diabetes. [0003] The incidence of tRNA Leu(UUR)3243A>G mutation in diabetic patients can reach more than 50%, a...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/156
Inventor 于超计吴文立王倩玉魏星
Owner 北京华诺奥美基因生物科技有限公司
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