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Special primer for simultaneously and accurately detecting SNP loci of related genes of 'slap-induced deafness' and ' injection-induced deafness'

A deafening and slapping technology, which is applied in the direction of recombinant DNA technology, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems that there is no application of matrix-assisted laser analysis ionization time-of-flight mass spectrometry technology, and achieve good accuracy , cost saving, high application value effect

Active Publication Date: 2021-02-26
中国人民解放军联勤保障部队第九八四医院
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry has not yet been applied in this field

Method used

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  • Special primer for simultaneously and accurately detecting SNP loci of related genes of 'slap-induced deafness' and ' injection-induced deafness'
  • Special primer for simultaneously and accurately detecting SNP loci of related genes of 'slap-induced deafness' and ' injection-induced deafness'
  • Special primer for simultaneously and accurately detecting SNP loci of related genes of 'slap-induced deafness' and ' injection-induced deafness'

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] This example is used to illustrate the special primers provided by the present invention for simultaneously and accurately detecting the SNP sites of genes related to "deafness caused by one slap" and "deafness caused by one needle".

[0096] 根据“一巴掌致聋”和“一针致聋”相关基因SNP位点:rs111033305、rs111033318、rs200455203、rs201562855、rs267606617、rs121908363、rs267606619、rs111033220、rs121908362、rs1057516953、rs111033380、rs192366176、rs111033313,设计特异性PCR Primers and single-base extension primers form a multiplex nested PCR reaction system to achieve "deafness with one slap" and "deafness with one needle" using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) technology (MASSARRAY platform). Deaf" accurate early warning detection.

[0097] Specific PCR primers and single base extension primers include:

[0098] Sequence 1, namely rs111033305 PCR primer 1: 5'-ACGTTGGATGTTGTTTTGTGGCCACCACTG-3'.

[0099] Sequence 2, namely rs111033305 PCR primer 2: 5'-A...

Embodiment 2

[0138] This example is used to illustrate the method provided by the present invention for simultaneously and accurately detecting the SNP sites of genes related to "deafness caused by one slap" and "deafness caused by one needle".

[0139] 1. Template whole genome DNA extraction

[0140] Genomic DNA was extracted from peripheral blood of 384 cases, and the concentration was uniformly diluted to 20-30ng / μL.

[0141] 2. PCR reaction

[0142] Prepare a reaction system in a 5 μL PCR 96-well plate: 1 μL template DNA, 1 μL amplification primer Mix, PCR buffer (containing 15 mM Mg 2+ )0.5μL, MgCl 2 (25mM) 0.4μL, dNTP (25mM) 0.1μL, Hotstar Taq (5U / μL) 0.2μL, make up to 5μL with MBG (Molecular Biology Grade) water. Seal the 384-well plate with Parafilm.

[0143]The PCR amplification primer Mix consists of primers shown in sequences 1-26 and water, wherein the concentration of each primer is 0.5 μM.

[0144] The above PCR reaction program is set as follows:

[0145]

[0146] 3...

Embodiment 3

[0172] This example is used to illustrate the method of detecting SNP sites of "one slap to deafness" and "one needle to deafness" and the application of special primers.

[0173] 1. Template DNA (whole genome) extraction

[0174] The peripheral blood of 8 subjects who were "deaf with one slap" was taken as samples 1-8 to be tested, and the peripheral blood of 8 subjects who were "deaf with one needle" was taken as samples 9-16 to be tested. 17-24 were normal control sample.

[0175] 2. PCR reaction: see step 2 of Example 2.

[0176] 3. Alkaline phosphatase treatment (SAP digestion reaction): see step 3 of Example 2.

[0177] 4. Single base extension reaction: refer to step 4 of Example 2.

[0178] 5. Resin purification: refer to Step 5 of Example 2.

[0179] 6. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry detection: see step 6 of Example 2.

[0180] After testing, in the 24 samples tested in this embodiment, the detection rate of each site...

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Abstract

The invention belongs to the technical field of biology, and provides a special primer for simultaneously and accurately detecting SNP loci of related genes of 'slap-induced deafness' and ' injection-induced deafness'. The special primer comprises a primer group 1 and a primer group 2, wherein the primer group 1 is a specific PCR primer composed of 13 pairs of primers, and the primer group 2 is asingle-base extension primer composed of 13 primers. The invention further provides a preparation for simultaneously and accurately detecting the SNP loci of the related genes of 'slap-induced deafness' and 'injection-induced deafness', and a method for simultaneously and accurately detecting the SNP loci of the related genes of 'slap-induced deafness' and 'injection-induced deafness'. By adoptingthe special primer provided by the invention, accurate prediction of high-risk groups of 'slap-induced deafness' and 'injection-induced deafness' can be efficiently completed simultaneously through one-time detection, the detection time is greatly shortened, the cost is saved, and the special primer has very high application value for preventing 'slap-induced deafness' and 'injection-induced deafness' and 'deafness-induced dumbness' caused by 'slap-induced deafness' and 'injection-induced deafness'.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a special primer for simultaneously and accurately detecting the SNP sites of genes related to "deafness caused by one slap" and "deafness caused by one needle" and its application. Background technique [0002] The incidence of deafness is relatively high in our country. The latest research results show that the proportion of genetic mutations that cause genetic deafness among the Chinese population at this stage is about 12%, that is, 12 out of every 100 people carry genetic defects that can cause genetic deafness. In 2008, 2009 and 2010, the incidence rate of congenital deafness in my country was 1.99‰, 2.15‰ and 2.19‰, that is, there were 2-3 deaf children in 1000 newborns, and more than half of the newborn deaf children were caused by Hereditary deafness caused by a mutation in the deaf gene. [0003] Individuals carry different gene single nucleotide polymorphisms,...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6883C12Q1/6858C12Q2600/156C12Q2531/113C12Q2521/525C12Q2533/101C12Q2565/627
Inventor 张鹏王瑛吕江宁
Owner 中国人民解放军联勤保障部队第九八四医院
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