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Non-syndromic deafness gene polymorphism detecting kit and application thereof

A technology for deafness genes and syndromes, which is applied in the determination/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of long detection time, acceptance, and expensive detection costs, and achieve simple operation, low cost, The effect of small sample size

Inactive Publication Date: 2016-03-30
CHANGSHA DIAN MEDICAL SCI INSPECTION CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, genetic testing for deafness has been carried out using gene chip technology in China, but its expensive testing costs cannot be accepted by the general public
The first-generation sequencing method Sanger sequencing can also detect deafness genes, but it also has the disadvantages of long detection time and high detection cost

Method used

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  • Non-syndromic deafness gene polymorphism detecting kit and application thereof
  • Non-syndromic deafness gene polymorphism detecting kit and application thereof
  • Non-syndromic deafness gene polymorphism detecting kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Reagents.

[0054] (1) DNA extraction reagents:

[0055] Purchased from QIAGEN Company.

[0056] (2) PCR reaction solution:

[0057] Amplification primers SEQIDNO: 1-10, synthesized by Shanghai Handsome Biotechnology Co., Ltd.;

[0058] PCRBuffer: purchased from Fermentas, USA;

[0059] MgCl 2 : purchased from the U.S. Fermentas company;

[0060] dNTPs: purchased from Fermentas, USA;

[0061] TaqDNA polymerase: purchased from Fermentas, USA.

[0062] (3) Reagent for single-strand purification:

[0063] 75% (v / v) ethanol solution: purchased from Hangzhou Changzheng Chemical Reagent Co., Ltd.;

[0064] 0.2mol / LNaOH: purchased from Shanghai Shisi Hewei Chemical Co., Ltd.;

[0065] 10mM Tris-Acetate (pH7.6): Tris-base was purchased from Sigma, USA, and anhydrous acetic acid was purchased from Hangzhou Chemical Reagent Co., Ltd.;

[0066] Binding buffer: 10mM Tris-HCl (Tris-base was purchased from Sigma, USA; hydrochloric acid was purchased from Hangzhou...

Embodiment 2

[0074] Embodiment 2: detection method.

[0075] Instruments: Bio-RadS1000PCR instrument, BeckmanMicrofuge22R desktop microrefrigerated centrifuge, Beijing Liuyi agarose gel electrophoresis instrument, Shanghai Peiqing gel imaging system, QIAGENPyroMarkQ96ID sequencer.

[0076] (1) Extract the DNA in the whole blood sample, the steps are as follows:

[0077] a. The preparation and inspection of reagent materials before the experiment are as follows: check the shelf life of the kit and ensure that ethanol has been added to WashBuffer 1 and 2, and tick the corresponding mark on the bottle; isopropanol (if not available, absolute ethanol can be used instead) and 75% ethanol; 1.5mL Eppendorf tubes and various pipette tips within the expiry date of autoclaving.

[0078] b. Take out the EDTA anticoagulant tube containing whole blood from the refrigerator at 4°C, and mix it upside down several times;

[0079] c. Pipette 900uL of cell lysate into a sterilized 1.5mL Eppendorf tube;

...

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Abstract

The invention discloses non-syndromic deafness gene polymorphism detecting primers. The primers mainly include five pairs of specific primers and nine sequencing primers for amplification of deafness genes, GJB2 genes, GJB3 genes, SLC26A4 genes and 12SrRNA genes are included, and ten single nucleotide polymorphisms are involved, namely GJB2 (35DELG, 176-191DEL16, 235DELC, 299-300 DEL AT), GJB3 (538C>T, 547G>A), SLC26A4(2168A>G, IVS7A>G) and mitochondria 12SrRNA (1494C>T, 1555A>G). The invention further discloses a kit comprising the primers. Sensitivity is high, results are visual, judgment is more accurate and quicker, and accurate and quick high-pass non-syndromic deafness gene polymorphism detection can be achieved.

Description

technical field [0001] The invention belongs to the technical field of molecular diagnosis, and in particular relates to a kit for detecting non-syndromic deafness gene polymorphism by pyrosequencing and an application thereof. Background technique [0002] Deafness is a group of diseases with high clinical heterogeneity. The age of onset, severity and damage location are different. More than 60% of deafness is related to genetic factors, and 80% of them are non-synthetic deafness (NSHI) without other clinical symptoms. ). The Human Genome Project has greatly promoted the research on the etiology of hereditary deafness. In recent years, scientists at home and abroad have found through a large number of studies that most hereditary deafness is a single-gene disease. So far, more than 400 syndromes with hearing impairment have been discovered, and more than 30 related genetic defects have been identified. In non-synthetic hereditary deafness, more than 100 pathogenic sites ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q2531/113C12Q2535/122C12Q2565/301
Inventor 任绪义虞闰六施宏
Owner CHANGSHA DIAN MEDICAL SCI INSPECTION CO LTD
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