Standard substance and kit for detecting mitochondrial A3243G heterozygous mutation rate and detection method

A technology of mitochondria and standard products, applied in the field of biomedical engineering, can solve the problems of insufficient sensitivity and lack of quantitative detection methods for mitochondria

Active Publication Date: 2013-12-18
SHANGHAI CHILDRENS HOSPITAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The technical problem to be solved by the present invention is to provide a more sensit

Method used

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  • Standard substance and kit for detecting mitochondrial A3243G heterozygous mutation rate and detection method
  • Standard substance and kit for detecting mitochondrial A3243G heterozygous mutation rate and detection method
  • Standard substance and kit for detecting mitochondrial A3243G heterozygous mutation rate and detection method

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Experimental program
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Embodiment 1

[0068] The preparation of embodiment 1 standard substance

[0069] 1. Extract DNA from the peripheral blood of patients confirmed to be A3243G mutations and non-mutated normal people, and use QIAGEN's peripheral blood DNA extraction kit for extraction.

[0070] 2. Using amplification primers for PCR amplification, the amplification product includes the site where the A3243G mutation is located;

[0071] The primer sequences are:

[0072] MT3243-F (SEQ ID NO.3 in the sequence listing):

[0073] 5'-AGAACAGGGTTTGTTAAGATGGC-3';

[0074] MT3243-R (SEQ ID NO.4 in the sequence listing):

[0075] 5'-GTTTTAAAGTTTTATGCGATTACCG-3'

[0076] The 5' of primer MT3243-R was labeled with biotin.

[0077] The PCR amplification program is:

[0078] ①94°C, 5min; ②94°C, 20s; ③55°C, 20s; ④72°C, 20s; the cycle number of steps ② to ④ is 40; ⑤72°C, 10min.

[0079] 3. Load the PCR products into the pGEM-T vector respectively. Because the end of the PCR product has a protruding A, and the pGEM-T ...

Embodiment 2

[0099] The application of embodiment 2 detection standard substance and regression equation in detection

[0100] After the standard product and regression equation were prepared, we applied it to the quantitative detection of the mitochondrial A3243G heterozygous mutation rate in the peripheral blood samples of patients with early-onset diabetes.

[0101] 1. Method

[0102] 1, extract DNA from patient's peripheral blood, method is the same as embodiment 1;

[0103] 2. Use the method of gel electrophoresis to detect the quality of the extracted DNA, and use a spectrophotometer to measure the concentration of the DNA;

[0104] 3. Dilute the detected DNA to 50ng / μL with TE;

[0105] 4. Using amplification primers MT3243-F and MT3243-R to pair, amplify the DNA sample of the patient, and the amplification procedure is the same as in Example 1;

[0106] 5. After the PCR amplification product is identified by electrophoresis, pyrosequencing is carried out using the pyrosequencing...

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Abstract

The invention discloses a standard substance and a kit for detecting mitochondrial A3243G heterozygous mutation rate and a detection method. The standard substance for detecting mitochondrial A3243G heterozygous mutation rate provided by the invention comprises: (1) a vector containing a mitochondrial DNA fragment with 3243 locus being a wild type A; and (2) a vector containing a mitochondrial DNA fragment with 3243 locus being a mutant G. The plasmid molecule standard substance, the detection method and a regression equation used for detecting data correction provided by the invention can conveniently and accurately detect A3243G heterozygous mutation rate in the mitochondrial DNA, and have advantages of high efficiency and sensitivity. In addition, the plasmid molecule standard substance, the detection method and the regression equation can accurately and quantificationally detect heterozygous mutation with mutation rate lower than 5%, which is a unique advantage unachievable by other heterozygous mutation detection means. The invention also has good reference meaning for quantitative determination of other heterozygous mutation in scientific research and production practice.

Description

technical field [0001] The invention relates to the field of biomedical engineering, in particular to a standard product, a kit and a detection method for detecting the mitochondrial A3243G heterozygous mutation rate. Background technique [0002] The A3243G mutation is the most common pathogenic mutation on mitochondrial genomic DNA (mito-chondrial DNA, mtDNA), which often exists in a heterozygous form, that is, there are both wild-type mtDNA and mutant mtDNA in a cell. A3243G mutation is one of the pathogenic factors of premature diabetes and neurological deafness, and the A3243G mutation rate is related to the severity of clinical symptoms. Therefore, the accurate quantitative detection of the A3243G mutation is of great significance. The mtDNA3243 site is located between 16S rRNA and tRNA Leu At the junction of the (UUR) gene, the study of the A / G mutation at the mtDNA3243 site found that the A base is located on the dihydrourinophilic loop of the secondary structure o...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/63C12N15/64
Inventor 曾溢滔颜景斌曾凡一方彧聃
Owner SHANGHAI CHILDRENS HOSPITAL
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