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A primer and kit for detecting cpt-ii gene mutation

A CPT-II, kit technology, applied in the direction of determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc.

Inactive Publication Date: 2016-02-17
QILU CHILDRENS HOSPITAL OF SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The detection of CPT-II gene mutation is usually for a certain hotspot mutation, and there is no report on the simultaneous detection of multiple mutations of CPT-II gene using multiple specific primers

Method used

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  • A primer and kit for detecting cpt-ii gene mutation
  • A primer and kit for detecting cpt-ii gene mutation
  • A primer and kit for detecting cpt-ii gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Design of Specific Amplification Primers

[0050] Design specific amplification primers according to the reference sequence of the CPT-II normal gene in the NCBI database, and the specific amplification primers should meet the following conditions:

[0051] (1) The product cannot form a secondary structure, the bases should be randomly distributed, and the primer itself cannot have 4 consecutive bases complementary;

[0052](2) The annealing temperature difference between the forward and reverse primers of all PCR fragments does not exceed 5°C, and the annealing temperature of each product is between 52°C and 58°C to ensure the specificity and stability of amplification;

[0053] (3) The forward amplification primer of the PCR amplification primer is a primer for sequencing amplification, and the length is between 18-20 bp.

[0054] The specific amplification primers designed in the present invention are shown in Table 1.

Embodiment 2

[0055] Embodiment 2: sample DNA extraction

[0056] The positive sample used in the present invention was taken from a subject in Shandong who was diagnosed with carnitine palmitoyltransferase II deficiency by the hospital, and the blood sample was taken with the consent of the subject.

[0057] Using the blood genome extraction kit (Tiangen Biochemical Technology [Beijing] Co., Ltd.) to extract genomic DNA, the exemplary steps are as follows:

[0058] (1) Take 900 μl of erythrocyte lysate and put it into a 1.5mL EP tube;

[0059] (2) Add 300 μL of whole blood to the above EP tube, mix upside down, and incubate at room temperature for 10 minutes to lyse red blood cells. Note that during the incubation period, mix upside down at least once;

[0060] (3) Centrifuge at 13200×g for 20 s at room temperature, and remove most of the supernatant with a tip (note that about 20 μL-30 μl of the supernatant is left in the tube);

[0061] (4) Vortex the upper tube to suspend the cells in...

Embodiment 3

[0072] Embodiment 3: PCR amplification

[0073] Using the specific amplification primers designed in Example 1, PCR amplification was carried out using sample DNA as a template. The reaction system for PCR amplification was 50 μL, and the specific composition was: 3 μL of DNA template, each primer (forward and reverse) 2 μL, Taq enzyme 0.4 μL, dNTP 4 μL, 10×PCR buffer 5 μL, double distilled water to make up to a total volume of 50 μL.

[0074] PCR amplification conditions were as follows: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 35 s, annealing for 35 s, annealing temperature for specific amplification primers at 52.8-57°C, extension at 72°C for 50 s, a total of 30 cycles; final extension at 72°C for 5 min.

[0075] The annealing temperature of the specific amplification primers was investigated and optimized, and the optimized annealing temperatures of each specific amplification primer are shown in Table 1.

[0076] Table 1. Specific amplification primer...

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Abstract

The invention discloses a primer for detecting CPT-II gene mutation. The primer comprises mutational primers for amplification of all exons of CPT-II genes and the junctional zone of exons and introns, and the primer sequence is shown as SEQ ID NO.1-SEQ ID NO.16. The invention further discloses a reagent kit for detecting CPT-II gene mutation and a method for detecting CPT-II gene mutation for non-diagnosis purpose. By the primer, the reagent kit and the method for detecting CPT-II gene mutation, the limit of detection of hotspot mutation in the prior art is overcome, all exons and the exon / intron junctional zone of the CPT-II genes can be captured by the specially designed amplification primer, and multiple potential mutational sites can be detected at one time.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a primer, method and kit for detecting CPT-II gene mutation. Background technique [0002] Carnitine Palmitoyltransferase Ⅱ (Carnitine Palmitoyltransferase-Ⅱ, CPT-Ⅱ) is located on the inner membrane of the mitochondria of cells and is one of the enzymes that play a key role in the oxidation of fatty acids. It reversibly catalyzes the transfer of acyl groups from acyl-CoA to L- The carnitine response plays an important role in the transport of long-chain fatty acids from the outer mitochondrial membrane to the inner mitochondrial membrane. The CPT-II gene is 3090 nucleotides long, located on chromosome 1p32, contains 5 exons and 4 introns, and encodes a 658 amino acid peptide chain. Mutations in the CPT-II gene cause CPT-II functional defects, and the function of the carnitine-dependent transport system will be destroyed, resulting in the inability to effectively convert fatty acylca...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 王广新马衍辉王大伟伊长英
Owner QILU CHILDRENS HOSPITAL OF SHANDONG UNIV
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