Spinal muscular atrophy detection kit and application thereof

A spinal muscular atrophy, detection kit technology, applied in the direction of microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of difficult clinical application, inability to detect carriers, and rising reagent costs, and achieve detection. The time required is short, the cost of inspection is reduced, and the effect of rapid inspection

Inactive Publication Date: 2018-10-26
广东辉锦创兴生物医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PCR-RFLP can only detect the case of homozygous deletion of exon 7, but cannot detect carriers with 1 copy
MLPA can detect the homozygous deletion of exon 7, the carrier with 1 copy, and the normal person with 2 copies, but the operation is complicated, the reagents are expensive, and it is not easy to promote clinical application
At present, there are also some commercial kits to quantitatively detect the change of SMN1 gene copy number by fluorescence quantitative method, but for the situation of distinguishing 1 copy and 2 copies, the fluorescence quantitative method first needs to make a standard curve, which makes clinical Reagent costs increase significantly when testing a small number of samples

Method used

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  • Spinal muscular atrophy detection kit and application thereof
  • Spinal muscular atrophy detection kit and application thereof

Examples

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Comparison scheme
Effect test

Embodiment 1

[0024] Example 1. Spinal muscular atrophy detection kit

[0025] In order to overcome the defects of the method for quantitatively detecting the copy number of exon 7 (ex7) of the SMN1 gene mentioned above, the present invention provides a kit for absolute quantification of the copy number of the SMN1 gene ex7 based on a droplet digital PCR method. The primers, probe sequences, and digital PCR reaction system used in the method are as follows.

[0026] Table 1. Primers and probes for digital PCR detection of SMN1 gene ex7

[0027] name

Sequence (5'-3')

serial number

SMN1-F

AAATGTCTTGTGAAACAAAATGC

SEQ ID NO:1

SMN1-R

GAATGTGAGCACCTTCCTTCT

SEQ ID NO:2

SMN1-P

CCGCTTCATCGCAGTGGGCTACGTG

SEQ ID NO:3

[0028] In order to monitor whether the qPCR reaction system is working normally, the internal reference gene human RPP30 gene was added at the same time, and the primers and probes of the RPP30 gene are shown in Table 2...

Embodiment 2

[0033] Example 2. Application of Spinal Muscular Atrophy Detection Kit

[0034] For clinical samples of peripheral whole blood, the sample type detected by the present invention, the extracted genomic DNA template is diluted to 20 ng / μl. After the genomic DNA was extracted from the blood sample, the ex7 deletion of SMN1 was detected by MLPA to find ex7 wild type, heterozygous type with 1 copy number deletion, and homozygous deletion type samples with 2 copy number deletions.

[0035] The microdroplet digital PCR reaction includes four steps: preparation system, microdroplet generation, amplification cycle and signal reading.

[0036] 1. Instruments and consumables: droplet generation instrument, PCR instrument T-100, oil droplet reader, PX1 heat sealer, droplet generation consumables (including DG8 cartridge, holder, rubber pad gasket), tin sealing film, 96 The orifice plates were all from Bo-Rad, USA.

[0037] 2. PCR reagents were purchased from Beijing Kangwei Century Comp...

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Abstract

The invention relates to a spinal muscular atrophy detection kit. The kit comprises a reagent which is used for quantitatively detecting the genotype of the 7th exon of an SMN1 gene by a droplet typedigital PCR method, wherein the reagent is a reagent for quantitatively detecting the copy number of the 7th exon of an SMN1 gene based on the droplet type digital PCR method. The kit has high exon detection accuracy, good repeatability, no false positive or false negative, high sensitivity and short detection time.

Description

technical field [0001] The invention relates to the field of molecular diagnosis, in particular to a detection kit for spinal muscular atrophy and its application. Background technique [0002] Spinal muscular atrophy (SMA) is one of the most common autosomal recessive neuromuscular diseases in the world. The gene carrier frequency in our population is about 1 / 50. The main clinical features of SMA are progressive anterior horn motor neuron degeneration and proximal muscle atrophy. There is currently no standardized medical intervention and clear treatment drugs for SMA patients, and children with severe illness often die of respiratory failure. Due to the poor prognosis of SMA, there is currently no effective treatment, so preventing the birth of children with this disease through genetic screening and prenatal diagnosis is the most effective way to prevent this disease. [0003] The SMA causative gene is the motor neuron survival gene (suvival motor neuron, SMN) located ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851
CPCC12Q1/6851C12Q2563/159C12Q2537/16C12Q2545/101
Inventor 胡锦张凤笑
Owner 广东辉锦创兴生物医学科技有限公司
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