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Genetic diagnosis kit for amyotrophic lateral sclerosis

A technology for lateral sclerosis and genetic diagnosis, applied in the field of molecular biology, can solve the problems of patents and reports of molecular diagnosis of ALS diseases that have not yet been seen, and achieve great popularization and application value

Active Publication Date: 2014-07-02
江苏雄鸣医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Retrieval of relevant materials including Chinese patents shows that RT-PCR developed on the basis of PCR combined with the now-developed gene sequencing technology can simultaneously detect multiple exons of a disease-causing gene in ALS in a single tube. Mutations, no patents or reports on the molecular diagnosis of ALS disease with this technology

Method used

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  • Genetic diagnosis kit for amyotrophic lateral sclerosis
  • Genetic diagnosis kit for amyotrophic lateral sclerosis
  • Genetic diagnosis kit for amyotrophic lateral sclerosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Gene determination, primer design, kit composition and amplification procedure

[0039] The 11 genes involved in the present invention are related to ALS disease, and the correlation has been reported in literature. The sequences of 11 genes can be obtained through the following ways.

[0040] The gene sequence can be retrieved at http: / / www.ncbi.nlm.nih.gov / according to the following gene ID

[0041] Table 2 Gene ID

[0042]

[0043] In the process of primer design, it is mainly designed according to the conserved region of each gene, and the entire CDS region of each gene is specifically amplified by using primers. The forward and reverse primers are respectively designed in the 5'-UTR and 3'-UTR regions to ensure that the amplified product can cover all CDS regions, enabling the kit to detect mutation sites on all CDS regions.

[0044] Taking the PFN1 gene as an example, the gene sequence obtained after downloading is shown below (SEQ ID NO.23), wh...

Embodiment 2

[0057] Example 2 Detection of genetic diagnosis of amyotrophic lateral sclerosis

[0058] The gene diagnosis kit for familial amyotrophic lateral sclerosis (ALS) provided by the present invention can detect samples such as blood, body fluid, hair, bone, cell or tissue samples of the individual to be tested. In this example, the same standard cell line as in Example 1 was used.

[0059] 1. Extraction of RNA from individual human fibroblast skin cells to be tested:

[0060] 1. Take the sample to be tested, add 1ml Trizol solution, mix on ice, mash and homogenate; stand on ice for 10min;

[0061] 2. Add 200μl chloroform to the homogenate, shake vigorously for 15s, and incubate at room temperature for 10min

[0062] 3. Centrifuge at 12000g / min at 4°C for 15min

[0063] 4. Take the aqueous phase (upper layer), add 500μl isopropanol, shake gently, and incubate at room temperature for 10~15min

[0064] 5. 4°C, 12000g / min, l centrifuge for 10min

[0065] 6. Discard the supernatan...

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Abstract

The invention discloses a genetic genetic diagnosis kit for amyotrophic lateral sclerosis. The kit and a detection method are characterized in that gene-specific primers are designed and synthesized according to a virulence gene which is studied and reported to be correlated with the amyotrophic lateral sclerosis, including SOD1, FUS, VAPB, ANG, TARDBP, FIG4, OPTN, VCP, Sigmar1, CHMP2B and PFN1 sites, a sample is obtained from an individual to be detected, a RT-PCR technology is carried out, the RT-PCR product is subjected to sequencing analysis, next, the sample sequence is compared with a normal gene sequence to determine whether the sample sequence has disease-causing mutations. The kit is capable of detecting all disease-causing mutation sites of the coding sequence (CDS) of the virulence gene at a time, and has the advantages of being simple and quick, reliable in preparation, good in specificity, high in sensitivity and the like; the kit is capable of detecting the ALS patients and asymptomatic patients.

Description

[0001] technical field [0002] The invention relates to a gene diagnosis kit for hereditary diseases, more specifically a gene diagnosis kit for amyotrophic lateral sclerosis, belonging to the technical field of molecular biology. [0003] Background technique [0004] Amyotrophic lateral sclerosis (ALS) is a major type of motor neuron disease (MND), first described by Charcot in 1865. The lesions mainly involve the anterior horn cells of the spinal cord, brainstem motor nuclei, and pyramidal tracts, and are characterized by simultaneous damage to upper and lower motor neurons, which is a chronic progressive degenerative disease. The clinical manifestations of the patient are symptoms of simultaneous involvement of upper and lower motor neurons, muscle weakness, muscle atrophy, muscle fasciculation, spasm, active tendon reflexes, and pathological reflexes in the affected limbs. Involvement of upper medullary motor neurons can lead to spastic dysarthria, manifested as slo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2531/113
Inventor 陈实黎寒
Owner 江苏雄鸣医药科技有限公司
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