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Detection method and kit for spinal muscular atrophy related gene mutation

A spinal muscular atrophy and detection method technology is applied in the field of detection of spinal muscular atrophy-related gene mutations, which can solve the problems of long detection period, inability to judge diseases, and expensive instruments, and achieve rapid detection, ingenious design, and sample saving. Effect

Active Publication Date: 2015-06-03
CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the existing SMA detection methods use multiple ligation probe amplification method or gene sequencing method, which have the disadvantages of long detection cycle and expensive equipment, and it is impossible to judge the disease and its prognosis through one detection

Method used

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  • Detection method and kit for spinal muscular atrophy related gene mutation
  • Detection method and kit for spinal muscular atrophy related gene mutation
  • Detection method and kit for spinal muscular atrophy related gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The design of embodiment 1 primer and probe

[0037] Design primers and probes according to the gene sequences of SMN, NAIP, GTF2H2 and the internal reference gene GAPDH to ensure that the concentration of upstream and downstream primers is consistent to generate double-stranded DNA through PCR reaction; the probe is complementary to the single-stranded target of PCR amplification during the annealing period of PCR reaction The DNA is hybridized and hydrolyzed by the 5' exonuclease activity of Taq DNA polymerase. After hydrolysis, the FAM fluorescein is away from the 3' quenching group, and the FAM fluorescein generates a fluorescent signal under the excitation light. The sequences of the designed primers and probes are shown in Table 1, and they were synthesized by China Invitrogen Company.

[0038] Table 1 Sequences of primers and probes

[0039]

[0040]

Embodiment 2

[0041] The extraction of the whole genome DNA of embodiment 2 samples to be tested

[0042] Whole blood DNA was extracted using TIANGEN "Whole Blood Genomic DNA Extraction Kit".

[0043] The final volume of extracted DNA is 100ul, the concentration is above 30ng / ul; OD260 / 280 is between 1.8-2.0.

Embodiment 3

[0044] Example 3 Detection of 4 sites of the three related genes SMN, NAIP and GTF2H2 in spinal muscular atrophy

[0045] (1) Establishment of PCR reaction system

[0046] The primers and probes synthesized in Example 1 were taken, and the whole genome DNA extracted in Example 2 was used as a template, and PCR reaction system I and PCR reaction system II were established according to the data shown in Table 2.

[0047] Table 2 PCR reaction system

[0048]

[0049] In the above system, when performing PCR test, it is often necessary to add buffer, dNTP, DNase, primer, template, water and other reagents required for various PCR reactions to the PCR tube, and it is easy to operate if these reagents are not standardized. Contamination causes inaccurate PCR results. Therefore, in order to simplify the repeated addition of various reagents, buffer, dNTP, DNA Polymerase and other reagents are first mixed together, that is, prepared into a Master-mix and then used, which simplifie...

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Abstract

The invention relates to a detection method and a kit for spinal muscular atrophy related gene mutation. The detection method comprises the following steps: extracting whole genome DNA of an object; carrying out PCR amplification on the obtained whole genome DNA by virtue of a PCR primer pair I, a PCR primer pair II, a PCR primer pair IIII and a PCR primer pair IV, so as to obtain a PCR amplification product; analyzing the obtained PCR amplification product, and judging whether four sites of spinal muscular atrophy related genes in the whole genome DNA of a to-be-detected sample. The kit comprises the PCR primer pair I, the PCR primer pair II, the PCR primer pair IIII and the PCR primer pair IV. The detection method and the kit have the beneficial effects that the design is smart, the sample is saved, meanwhile, three genes can be detected, the detection is rapid, accurate, simple and convenient, and a detection result can be taken as a reference basis of diagnosis, treatment and medication of physicians; the detection method and the kit are applicable to large-scale popularization and application.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a detection method, kit and application of spinal muscular atrophy-related gene mutations. Background technique [0002] Progressive spinal muscular atrophy (SMA), also known as spinal muscular atrophy or spinal muscular atrophy, is a type of disease that causes muscle weakness and muscle atrophy due to the degeneration of motor neurons in the anterior horn of the spinal cord and the motor nucleus of the brainstem. Spinal muscular atrophy is a common autosomal recessive genetic disease of the nervous system, ranking second among fatal autosomal recessive genetic diseases, with an incidence rate of 1 / 6000-1 / 10000 among live births. The gene carrier frequency of the disease is 1:40-1:60. SMA can be divided into 3 types: SMAI type, also known as infantile severe, Werdnig-Hoffmann disease, children with onset within 6 months after birth, generalized severe muscle weakness and decreased...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 刘之岱邹琳何晓燕唐诗刘姗蒋莉
Owner CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV
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