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Reagent kit for detecting 387delC mutation of large vestibular aqueduct related gene SLC26A4

A technology of SLC26A4 and kit, which is applied in the direction of recombinant DNA technology, measurement/testing of microorganisms, DNA/RNA fragments, etc., and can solve problems such as changes in membrane labyrinth structure

Inactive Publication Date: 2009-07-29
GENERAL HOSPITAL OF PLA
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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 kit for detecting 387delC mutation of large vestibular aqueduct related gene SLC26A4
  • Reagent kit for detecting 387delC mutation of large vestibular aqueduct related gene SLC26A4
  • Reagent kit for detecting 387delC mutation of large vestibular aqueduct related gene SLC26A4

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] [Example 1] Detection method for 109G>T heterozygous mutation of SLC26A4 gene

[0065] 1. Preparation of blood sample DNA of the subject to be tested

[0066] 1. Research objects: 89 patients with enlarged vestibular aqueduct collected from the deaf outpatient clinic, and 80 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.

[0067] 2. Genomic DNA extraction: using phenol-chloroform extraction method.

[0068] first day

[0069] 1) Anticoagulant blood was diluted 1-fold with PBS.

[0070] 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 ...

Embodiment 2

[0148] [Example 2] Research on the evolution of mutation sites

[0149] Mutation analysis: sequence comparison analysis was performed using SeqmanTM software in the DNAStar5.0 (Lasergene Inc.) software package. The sequence obtained by sequencing was compared with the standard sequence retrieved by NCBI to find out the mutant sequence and the mutation site (109G>T, E37X).

[0150] Evolutionary study: The 109G>T mutation is located at the amino terminal of Pendrin, which terminates the amino acid code at position 37, and the subsequent 734 amino acids are lost. This mutation must affect the structure and function of Pendrin.

[0151] Detection of 387delC heterozygous mutation in SLC26A4 gene

[0152]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 89 family members of the disease and 80 normal controls, it was found that a patient with an enlarged vestibular aqueduc...

Embodiment 3

[0153] [Example 3] Detection method for 387delC heterozygous mutation of SLC26A4 gene

[0154] See Example 1 for the basic method and steps. In the specific method, PCR amplification is performed on the exon 4 coding region of the SLC26A4 gene and its flanking sequences. The PCR primer sequences used are:

[0155] Upstream primers:

[0156] PDS4-F: 5'-TAATCACTTTGCATGTGCTTT-3'(nt11480-nt11500)

[0157] Downstream primers:

[0158] PDS4-R: 5'-GCCAAAACACTTTAAACATGA-3'(nt11648-11nt668)

[0159] Note: SLC26A4 gene sequence retrieval number: GeneID: 5172

[0160] 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: Figure 8 shown. The agarose gel electrophoresis patterns of electrophoresis and quantification of PCR products are shown in Figure 9 ; Figure 10 shown.

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Abstract

The invention relates to a detection method. In the method, whether SLC26A4 gene mutation exists in a sample from an individual to be detected is detected to diagnose on occurrence and type of a large vestibular aqueduct disease in the individual to be detected, the SLC26A4 gene mutation is 387delC heterozygous mutation located at an SLC26A4 gene exon 4. The invention also relates to a detection kit used for detecting whether SLC26A4 gene mutation exists in the sample from the individual to be detected and application of SLC26A4 gene mutation in diagnosis and / or treatment of diseases related to large vestibular aqueduct. The gene mutation and detection method are beneficial to clinically carrying out screening of SLC26A4 gene mutation of people suffering from deafness, thus providing service for diagnosis and treatment of the people suffering from deafness.

Description

[0001] This application is a divisional application of Chinese patent application 200510132214.5. 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 enlargeme...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 王秋菊赵亚丽
Owner GENERAL HOSPITAL OF PLA
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