Reagent case for detecting 349delC mutation of large vestibular aqueduct related gene SLC26A4

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 349delC heterozygous mutation located at SLC26A4 exon 4. 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 200510087749.5. 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...

Examples

Embodiment 1

[0062] [Example 1] Detection method for 349delC heterozygous mutation of SLC26A4 gene

[0063] 1. Preparation of blood sample DNA of the subject to be tested

[0064] 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.

[0065] 2. Genomic DNA extraction: using phenol-chloroform extraction method.

[0066] first day

[0067] 1) Anticoagulant blood was diluted 1-fold with PBS.

[0068] 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

[0151] [Example 2] Research on the evolution of mutation sites

[0152] 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).

[0153]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.

[0154] Kit for detecting SLC26A4 gene mutation related to enlargement of vestibular aqueduct and its application

Embodiment 3

[0156] The test kit for detecting SLC26A4 gene mutation can be prepared as a separate test kit for detecting each single mutation site as required, or can be prepared as a test kit for detecting multiple or all SLC26A4 gene mutation sites, as follows to detect the mutation site of the present invention Point kit as an example for illustration:

[0157] 1. The kit contains:

[0158] (1) Primers for amplification

[0159] 1 pair of primers designed to detect the 349delC mutation site of the SLC26A4 gene, or other available primers designed for the mutant gene;

[0160] Note: SLC26A4 gene sequence retrieval number: GeneID: 5172

[0161] Other reagents optionally included in the kit include the following:

[0162] (2) Taq enzyme 5U / μl for PCR amplification

[0163] (3) 10× buffer solution (containing 15ml MgCl 2 )

[0164] (4)dNTP 2.5mM

[0165] (5) Eco31I restriction endonuclease; BseM I restriction endonuclease; Hind III restriction endonuclease; Nde I restriction endonuc...

[0001] This application is a divisional application of Chinese patent application 200510087749.5. 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...

Examples

Embodiment 1

[0062] [Example 1] Detection method for 349delC heterozygous mutation of SLC26A4 gene

[0063] 1. Preparation of blood sample DNA of the subject to be tested

[0064] 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.

[0065] 2. Genomic DNA extraction: using phenol-chloroform extraction method.

[0066] first day

[0067] 1) Anticoagulant blood was diluted 1-fold with PBS.

[0068] 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

[0151] [Example 2] Research on the evolution of mutation sites

[0152] 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).

[0153]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.

[0154] Kit for detecting SLC26A4 gene mutation related to enlargement of vestibular aqueduct and its application

Embodiment 3

[0156] The test kit for detecting SLC26A4 gene mutation can be prepared as a separate test kit for detecting each single mutation site as required, or can be prepared as a test kit for detecting multiple or all SLC26A4 gene mutation sites, as follows to detect the mutation site of the present invention Point kit as an example for illustration:

[0157] 1. The kit contains:

[0158] (1) Primers for amplification

[0159] 1 pair of primers designed to detect the 349delC mutation site of the SLC26A4 gene, or other available primers designed for the mutant gene;

[0160] Note: SLC26A4 gene sequence retrieval number: GeneID: 5172

[0161] Other reagents optionally included in the kit include the following:

[0162] (2) Taq enzyme 5U / μl for PCR amplification

[0163] (3) 10× buffer solution (containing 15ml MgCl 2 )

[0164] (4)dNTP 2.5mM

[0165] (5) Eco31I restriction endonuclease; BseM I restriction endonuclease; Hind III restriction endonuclease; Nde I restriction endonuc...

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