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Digital PCR kit for detecting spinal muscular atrophy and application thereof

A spinal muscular atrophy and kit technology, applied in the biological field, can solve the problem of not being able to detect SMN1 exons 7, 8 and SMN2 genes at the same time

Active Publication Date: 2021-03-19
TSINGHUA UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

qPCR is easy and fast to operate, but it needs to rely on the standard curve for quantification, and the existing kits cannot detect SMN1 exons 7, 8 and SMN2 genes at the same time
[0007] At present, there is no technical solution based on digital PCR that can detect SMN1 exon 7, 8 and SMN2 genes in one tube

Method used

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  • Digital PCR kit for detecting spinal muscular atrophy and application thereof
  • Digital PCR kit for detecting spinal muscular atrophy and application thereof
  • Digital PCR kit for detecting spinal muscular atrophy and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 SMN1 exon 7, 8 and SMN2 exon 7 primer probe screening

[0029] 1. SMN1 exon 7 and SMN2 exon 7 primer probe screening

[0030] 1. SMN1 exon 7 and SMN2 exon 7 primer probe design

[0031] The SMN1 and SMN2 gene sequences were downloaded from NCBI for sequence analysis. Since there is only one base difference between SMN1 exon 7 and SMN2 exon 7, probes specifically targeting SMN1 and SMN2 exon 7 were designed for the different bases of the two genes, and the two genes used the same A pair of primers for amplification. The following table is the candidate primer probe sequence:

[0032]

[0033]

[0034] 2. Synthesis, digestion and quantification of plasmid reference products

[0035] In order to verify the amplification effect of the primers and probes, the plasmid reference products containing the target sequence were synthesized by Jinweizhi Company, respectively containing SMN1 exon 7 and SMN2 exon 7 plasmids, and the insertion sequence was 300bp. T...

Embodiment 2

[0078] Example 2 Human blood sample SMA detection

[0079] 1. Blood sample collection and DNA extraction

[0080] Blood samples from normal people, SMA carriers (SMN1 heterozygous deletion) and SMA patients (SMN1 homozygous deletion) were collected, DNA in the blood samples was extracted using a DNA extraction kit, and measured using a Nanodrop 2000 spectrophotometer and a Qubit 3.0 (ThermoScientific) fluorescence detector For the amount and purity of DNA, dilute the DNA template to 10ng / μL. All DNA samples were stored at -80°C until use.

[0081] 2. Primers and probes

[0082] In this experiment, five different gene sequences were amplified, SMN1 exon 7, SMN1 exon 8, SMN2 exon 7, internal reference gene RPS27A and internal reference gene RPP30. The 5' end of the probe is labeled with 5 different fluorophores, and the excitation and emission wavelengths of each fluorophore are independent to avoid mutual interference. This experiment uses FAM, VIC, ROX, Cy5 and Cy5.5 Fluor...

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Abstract

The invention provides a digital PCR kit for detecting spinal muscular atrophy. The digital PCR kit is a digital PCR kit for simultaneously detecting an SMN1 exon 7, an SMN1 exon 8, an SMN2 exon 7 andan internal reference gene in a sample to be detected in a one-tube digital PCR reaction, and the SMN1 exon 7, the SMN1 exon 8, the SMN2 exon 7 and the internal reference gene are simultaneously detected in at least four fluorescence channels. The kit disclosed by the invention is based on a digital PCR technology, can be used for quantitatively detecting the copy numbers of the SMN1 exons 7 and8 and the SMN2 in one tube, and can be applied to SMA clinical definite diagnosis, carrier screening and disease typing.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a digital PCR kit for detecting spinal muscular atrophy and its application. Background technique [0002] The autosomal recessive disorder spinal muscular atrophy (SMA) is a severe neuromuscular disorder characterized by the degeneration of alpha motor neurons in the spinal cord, leading to progressive weakness and paralysis of proximal muscles. SMA is the most common fatal autosomal recessive disease after cystic fibrosis, with an incidence rate of 1 / 6000-1 / 10000 and a carrier rate of 1 / 40-1 / 60. [0003] SMA is caused by mutations or deletions in the SMN1 gene. The SMN1 gene of normal people has 2 copies of the gene, and 95% of SMA patients have a homozygous deletion of SMN1 exon 7 (0 copy), and most of them are exon 7 and 8 deleted at the same time, and only a few of them are only deleted Exon 7, there have also been reports of patients missing only exon 8. In the remaining 5% ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6851
CPCC12Q1/6883C12Q1/6851C12Q2600/156C12Q2600/166C12Q2531/113C12Q2545/101C12Q2563/107
Inventor 陈芊如王芳郭娜郭永祝令香杨文军
Owner TSINGHUA UNIV
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