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Time-of-flight mass spectrometry nucleic acid analysis method for detecting human spinal muscular atrophy gene mutation

A spinal muscular atrophy and human technology, applied in biochemical equipment and methods, microbial measurement/testing, DNA/RNA fragments, etc., can solve the problems of high cost, time-consuming detection, difficult large-scale population screening and technology promotion And other issues

Active Publication Date: 2019-11-19
GUANGZHOU DARUI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For a long time, the routine clinical detection of SMA mainly uses the multiple ligation-dependent probe amplification (Multiple Ligation-dependent Probe Amplification, MLPA) detection kit of the Netherlands MRC company. Carry out large-scale population screening and technology promotion

Method used

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  • Time-of-flight mass spectrometry nucleic acid analysis method for detecting human spinal muscular atrophy gene mutation
  • Time-of-flight mass spectrometry nucleic acid analysis method for detecting human spinal muscular atrophy gene mutation
  • Time-of-flight mass spectrometry nucleic acid analysis method for detecting human spinal muscular atrophy gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] Embodiment 1 A kind of human spinal muscular atrophy gene detection kit

[0106] 1. Composition

[0107] Nucleotide sequence as shown in SEQ ID NO: 1-22 amplification primer, nucleotide sequence as shown in SEQ ID NO: 23-35 mass spectrum extension probe primer, multiplex PCR reaction reagent, single base extension reaction reagent Reagents with de-dNTP mix.

[0108] Wherein, all the amplification primers are premixed as a PCR amplification primer mixture; all the mass spectrum extension probes are premixed as a primer extension primer mixture.

[0109] Wherein, the multiplex PCR reaction reagents include: PCR amplification primer mixture, multiplex PCR enzyme, extension reaction multiplex PCR buffer, magnesium chloride buffer, and dNTP mixture.

[0110] More preferably, the single base extension reaction reagent includes: extension buffer, extension termination mixture, reaction catalyzing enzyme, and extension primer mixture.

[0111] More preferably, the dNTP remov...

Embodiment 2

[0156] Example 2 Calculation of relative copy number of target gene

[0157] Taking two samples as an example, the method of using the formula for calculating the copy number given by the present invention is illustrated.

[0158]

[0159] The following table shows the peak areas obtained after each sample is tested according to the kit of Example 1, and further calculates the peak area ratio (peak area ratio=target gene peak area / reference gene peak area)

[0160] Table 12:

[0161]

[0162]The relative copy number of the reference gene of all specimens is maintained at 2, and the relative copy number of the target gene of the normal reference sample is 2. The relative copy number of the target gene of the tested sample can be calculated based on the peak area ratio of the reference sample.

[0163] *: The target gene copy number of the tested sample 1 = (0.43 / 0.44) × 2 = 1.955, that is, 2 copies;

[0164] The target gene copy number of the tested sample 2=(0.20 / 0.44)...

Embodiment 3

[0165] Example 3 Detection of known genotype samples

[0166] 1. Experimental method

[0167] 1. Sample source and type

[0168] SMA gDNA specimens with confirmed genotypes selected from the laboratory specimen bank and collected by cooperative medical institutions, the genotypes are SMN1 / SMN2=2 / 0, SMN1 / SMN2=0 / 2 and there are no NAIP, GTF2H2 and H4F5 genes 5 samples for each deletion (10 samples in total), 10 samples of SMN1 / SMN2=2 / 2 and no deletion of NAIP, GTF2H2 and H4F5 genes, dilute gDNA samples to 10-50ng / μL with sterile double distilled water spare. Three cases of plasmid DNA with c.5C>T, c.22dupA, c.275G>C, c.683T>A, c.819_820insT and c.830A>G mutations in SMN1 gene were diluted to 0.001 with sterile double distilled water -0.002pg / μL spare.

[0169] 2. Sample detection

[0170] Sample detection was performed using the kit of Example 1.

[0171] 2. Experimental results

[0172] The results are shown in Table 13. The results obtained by using the kit of Example ...

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Abstract

The invention discloses a time-of-flight mass spectrometry nucleic acid analysis method for detecting human spinal muscular atrophy gene mutation. A primer combination comprising an amplification primer and a mass spectrometry extension probe primer is utilized. According to the method, the copy numbers of sequences related to SMN1, SMN2, NAIP, H4F5 and GTF2H2 genes are quantitatively detected, and whether deletion, deletion number and multiple copies exist or not is analyzed, so that the clinical phenotype severity can be directly deduced; the method has good sensitivity, specificity, stability and accuracy, and effectively solves the technical bottleneck of false negative, false positive and the like; the operation is simple, the cost is relatively low, and the result is stable and reliable; the method is high in flux and low in cost, has general representativeness and universality, is easy to realize automatic and large-scale detection, and is suitable for large-scale population screening; genetic typing detection can be carried out on part of common SMN1 upper point mutations; and the requirements of large-scale population screening, prenatal diagnosis and conventional molecular diagnosis in current SMA prevention and treatment are met.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a time-of-flight mass spectrometry nucleic acid analysis method for detecting human spinal muscular atrophy gene mutations. Background technique [0002] Spinal muscular atrophy (SMA) is a group of common autosomal recessive genetic diseases, ranking second among fatal autosomal recessive genetic diseases, and the incidence rate of live births is 1 / 6000~ 1 / 10000. Clinically, SMA is generally divided into 4 types: type Ⅰ (also known as Werding-Hoffman disease) is the most serious subtype (severe type), accounting for about half of SMA patients, with acute onset and rapid progression, usually in the first 6 months of life SMA type II is chronic infantile type (intermediate type), usually onset within 7 to 18 months, and most of them can survive 10 to 20 years old; SMA type III (also known as Wohlfart-Ku gelberg-Welander disease) is juvenile type (mild type), and the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11C12Q1/6813
CPCC12Q1/6813C12Q1/6883C12Q2600/156C12Q2565/627
Inventor 杨学习周万军吴英松朱安娜李明徐惠玲李婕
Owner GUANGZHOU DARUI BIOTECH
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