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Spinal muscular atrophy-related gene mutation detection method, related detection probe composition and detection kit as well as related application

A spinal muscular atrophy and detection kit technology, applied in the field of disease-related gene mutation detection and spinal muscular atrophy-related gene mutation detection, can solve problems such as easy contamination, avoid sample contamination, rapid detection, and clever design Effect

Inactive Publication Date: 2014-05-14
SHANGHAI CHROMYSKY MEDICAL RES
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AI Technical Summary

Problems solved by technology

[0004] Under this background, the current SMA diagnosis is based on clinical features, electromyography, PCR-RFLP, PCR-DHPLC, capillary electrophoresis, and sequencing analysis, etc., requiring special biochemical analyzers and reagents, and the detection takes about 1 week. Open tube detection operation is easy to cause pollution

Method used

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  • Spinal muscular atrophy-related gene mutation detection method, related detection probe composition and detection kit as well as related application
  • Spinal muscular atrophy-related gene mutation detection method, related detection probe composition and detection kit as well as related application
  • Spinal muscular atrophy-related gene mutation detection method, related detection probe composition and detection kit as well as related application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: DNA preparation

[0025] 1) Preparation of normal human genomic DNA for testing

[0026] Take normal human blood filter paper, extract and prepare by Chlex100, and adjust the concentration of normal human genomic DNA to 10ng / μl.

[0027] 2) SMN1 normal genotype standard preparation

[0028] According to the sequences of SMN1 and internal reference gene β-actin, SMN1EXON7 (SEQ ID NO:1), SMN1EXON8 (SEQ ID NO:2) and β-actin (SEQ ID NO:3) were artificially synthesized. The concentration of the T vector containing the above fragments was adjusted to 1ng / ul as a 10-fold normal genotype template.

[0029] 3) SMN1 mutation deletion genotype standard preparation

[0030]The internal reference gene β-actin (SEQ ID NO:3) was artificially synthesized. The concentration of the T vector containing the above fragments was adjusted to 1ng / ul as a template for the 10-fold mutation deletion genotype.

Embodiment 2

[0031] Embodiment 2: Design and synthesis of SMN1 mutant gene amplification primers and MGB probes

[0032] Synthetic primers were designed according to SMN1EXON7 and SMN1EXON8, the SMN1 gene probe was labeled Fam, and the internal reference gene probe was labeled Vic. Verify that fluorescent quantitative PCR primers specifically amplify target gene fragments, and Taqman MGB site-specific probes report corresponding fluorescent signals. Artificially synthesized gene fragment-specific amplification primers and site-specific Taqman MGB reporter probes. See Table 1 and Table 2 for gene fragment-specific primers and site-specific Taqman MGB probe sequences (SEQ ID NO: 4 to SEQ ID NO: 12).

[0033] Table 1

[0034]

[0035]

[0036] Table 2

[0037]

Embodiment 3

[0038] Example 3: Normal or missing detection of SMN1 Taqman quantitative PCR gene

[0039] 1) exon7 gene verification

[0040] Reaction system: 2.5 μl of primer-probe mixture, 12.5 μl of 2-fold Multiplex PCR Mix (QIAGEN N.V., Germany), 2.5 μl of standard template, 5 μl of 5-fold Easy Buffer, ddH 2 O2.5 μl.

[0041] Quantitative PCR instrument: ABI7500 (Life Technologies, USA).

[0042] Reaction conditions: 95°C for 5 minutes; 94°C for 30 seconds—60°C for 30 seconds—72°C for 45 seconds, 40 cycles.

[0043] 2) exon8 gene verification

[0044] Reaction system: 2.5 μl of primer-probe mixture, 12.5 μl of 2-fold Multiplex PCR Mix (QIAGEN N.V., Germany), 2.5 μl of standard template, 5 μl of 5-fold Easy Buffer, ddH 2 O2.5 μl.

[0045] Quantitative PCR instrument: ABI7500 (Life Technologies, USA).

[0046] Reaction conditions: 95°C for 5 minutes; 94°C for 30 seconds—60°C for 30 seconds—72°C for 45 seconds, 40 cycles.

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Abstract

The invention provides a spinal muscular atrophy-related gene mutation detection method which comprises the following steps: extracting genome DNA (deoxyribonucleic acid) of a detection object; performing fluorescent quantitative PCR (polymerase chain reaction) on the genome DNA by adopting a combination of two PCR primer pairs and two Taqman MGB fluorescent probes respectively, wherein the PCR primer pairs are nucleotide sequences shown in SEQ ID NO:4 and SEQ ID NO:5 as well as SEQ ID NO:7 and SEQ ID NO:8 respectively, and nucleotide sequences of the Taqman MGB fluorescent probes are nucleotide sequences shown in SEQ ID NO:6 and SEQ ID NO:9 respectively; analyzing whether two loci of a spinal muscular atrophy-related gene in the genome DNA mutate according to a real-time amplification curve formed by fluorescent signals collected from the fluorescent quantitative PCR. The invention further relates to a related detection probe composition and a detection kit, as well as a related application. According to the method, the design is skilful, the closed pipe detection is adopted, the sample pollution can be effectively avoided, the detection is quick, accurate, easy and convenient, detection results can serve as references for diagnosis, treatment and drug use of doctors, and large-scale popularization and application are facilitated.

Description

technical field [0001] The present invention relates to the technical field of disease-related gene mutation detection, more specifically, to the technical field of spinal muscular atrophy-related gene mutation detection, in particular to a spinal muscular atrophy-related gene mutation detection method and a related detection probe composition And detection kits, and related applications. Background technique [0002] Spinal muscular atrophy (SMA) is a common autosomal recessive genetic disease of the nervous system. The main related gene SMN1 of SMA is located at 5q13.1. The main cause of this disease is the homozygosity of the SMN1 gene. missing. SMA can be divided into 3 types: SMA type I, also known as infantile severe, Werdnig-Hoffmann's disease, occurs within 6 months after birth, with severe generalized muscle weakness and decreased muscle tone, unable to sit without a support, often at 2 Died of respiratory muscle paralysis before the age of 12; SMA type II, also k...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q2561/101
Inventor 赵翊均周巍
Owner SHANGHAI CHROMYSKY MEDICAL RES
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