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Reagent case for detecting 916_917insG mutation of large vestibular aqueduct related gene SLC26A4

A kit and aqueduct technology, applied in the field of kits for detecting SLC26A4 mutation gene, can solve problems such as changes in the structure of the membrane labyrinth

Inactive Publication Date: 2008-09-03
GENERAL HOSPITAL OF PLA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And due to toxic mechanisms of altered osmotic pressure and altered composition lead to altered membrane labyrinth structure

Method used

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  • Reagent case for detecting 916_917insG mutation of large vestibular aqueduct related gene SLC26A4
  • Reagent case for detecting 916_917insG mutation of large vestibular aqueduct related gene SLC26A4
  • Reagent case for detecting 916_917insG mutation of large vestibular aqueduct related gene SLC26A4

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] [Example 1] Detection method for 349delC heterozygous mutation of SLC26A4 gene

[0058] 1. Preparation of blood sample DNA of the subject to be tested

[0059] 1. Research objects: 70 patients with enlarged vestibular aqueduct collected from the Deaf Clinic, and 85 normal controls. All participants were investigated in detail about their medical history and family history, and a physical examination was performed on them. The otological examination included otoscopy and audiological evaluation. After signing the informed consent form, 5-10ml blood samples were collected from each person.

[0060] 2. Genomic DNA extraction: using phenol-chloroform extraction method.

[0061] first day

[0062] 1) Anticoagulant blood was diluted 1-fold with PBS.

[0063] 2) Add 2 times the volume of lymphatic separation solution (18°C-28°C) into the centrifuge tube, spread a layer of 1 times the volume of diluted blood on top, centrifuge at 1000×g at room temperature for 20 minutes. ...

Embodiment 2

[0144] [Example 2] Research on the evolution of mutation sites

[0145] Mutation analysis: using Seqman in the DNAStar5.0 (Lasergene inc.) software package TM software for sequence comparison analysis. The sequence obtained by sequencing was compared with the standard sequence retrieved by NCBI to find out the mutant sequence and the mutation site (349delC, X125).

[0146] Evolutionary research: The 349delC mutation is located in the second transmembrane region of Pendrin, which causes a frame shift in the amino acid sequence from position 117, and then ends prematurely at amino acid position 125. This mutation must affect the structure and function of Pendrin.

[0147] Detection of 916 917insG heterozygous mutation in SLC26A4 gene

[0148] A patient with an enlarged vestibular aqueduct was used as the research object. Through the screening of the exons in the coding region of the SLC26A4 gene in 70 family members of the disease and 87 normal controls, it was found that a...

Embodiment 3

[0149] [Example 3] Detection method for 916_917insG heterozygous mutation of SLC26A4 gene

[0150] See Example 1 for the basic method and steps. In the specific method, PCR amplification is performed on the exon 7 coding region of the SLC26A4 gene and its flanking sequences. The PCR primer sequences used are:

[0151] Upstream primers:

[0152] PDS7-F: 5'-CATGGTTTTTCATGTGGGAAGATTC-3'(nt22454-nt22478)

[0153] Downstream primers:

[0154] PDS7-R: 5'-AATGGCAGTAGCAATTATCG-3'(nt22822-nt22841)

[0155] The PCR reaction was carried out on the 9700 thermal cycler of ABI company, and the reaction process (including temperature and time) was as follows: image 3 shown. The agarose gel electrophoresis patterns of electrophoresis and quantification of PCR products are shown in Figure 4 ; Sequencing diagram see Figure 5 shown.

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Abstract

The invention relates to a detection method, by which occurrence and type of vestibular aqueduct enlargement of individuals under test are diagnosed by detecting whether SLC26A4 genetic mutation exists in a specimen from individuals under test, wherein the SLC26A4 genetic mutation is a 916_917insG heterozygous mutation located at SLC26A4 exon 7. The invention also relates to a detection kit for detecting whether SLC26A4 genetic mutation exists in a specimen from individuals under test, and the application of SLC26A4 genetic mutation to diagnosis and / or therapy of diseases related to vestibular aqueduct enlargement. The genetic mutation and detection method are helpful to clinical screen of SLC26A4 genetic mutation of deafness patients, and provide service for diagnosis and therapy of the deafness patients.

Description

[0001] This application is a divisional application of Chinese patent application CN200510087749.5. technical field [0002] The invention relates to a kit for detecting SLC26A4 mutation gene. Background technique [0003] The SLC26A4 gene is located at 7q31, which was originally located by Everett et al., and found that the mutation of this gene can lead to an autosomal recessive genetic disease: Pendred syndrome, clinical manifestations of goitre, and combined vestibular aqueduct enlargement or Mondini malformation ( Enlarged vestibular aqueduct combined with cochlear hypoplasia) sensorineural deafness. Later, Usami et al. screened the SLC26A4 gene in 6 families with simple vestibular aqueduct enlargement, showing that the mutation of this gene can also lead to simple vestibular aqueduct enlargement, and its genetic mode is also recessive inheritance. It can be seen that the SLC26A4 gene mutation Can cause vestibular aqueduct enlargement - pure vestibular aqueduct enlarge...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 王秋菊沈岩翟所强赵亚丽
Owner GENERAL HOSPITAL OF PLA
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