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Kit for detecting c.1671_1673del G mutation of OTOF gene

A kit and gene technology, applied in the field of kits for detecting OTOF gene mutations

Active Publication Date: 2014-04-16
王秋菊 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In patients with TYPE I auditory neuropathy spectrum disorder, the lesion is deep in the auditory nerve, and there is currently no effective treatment

Method used

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  • Kit for detecting c.1671_1673del G mutation of OTOF gene
  • Kit for detecting c.1671_1673del G mutation of OTOF gene
  • Kit for detecting c.1671_1673del G mutation of OTOF gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] 【Example 1】 Extraction of blood samples to be tested and PCR amplification of OTOF gene coding region

[0066] 1. Preparation of blood sample DNA of the subject to be tested

[0067] 1. Research object

[0068] 17 sporadic patients with non-syndromic auditory neuropathy spectrum disorder and 100 normal hearing controls without family history were screened for OTOF gene according to the following method. Among the 17 patients, 1 patient with auditory neuropathy spectrum disorder was found to be a compound heterozygote for the c.1671_1673delG mutation and another mutation. No c.1671_1673delG mutation was found in the screening of 100 normal hearing persons.

[0069] 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 pe...

Embodiment 2

[0099] [Example 2] Purification and Quantification of PCR Amplified Product of Coding Region of OTOF Gene

[0100] 1. Purification of PCR products——96-well plate method

[0101] 1. Add 50 μl sterile water to the 96-well plate containing the PCR product and mix well.

[0102] 2. Transfer it to the Millipore purification plate, put it on the vacuum pump for about 3 minutes, and see that there is no water in the purification plate.

[0103] 3. Add 50 μl of deionized water to the purification plate again, and continue to filter until there is no water in the purification plate.

[0104] 4. Remove the purification plate from the vacuum pump, add 20 μl of deionized water to the plate, let it rest for 15 minutes, shake it for another 15 minutes, and then suck it into a new 96-well plate.

[0105] 5. Store in a -20°C refrigerator.

[0106] 2. Quantification by electrophoresis

[0107] 1. Sample preparation

[0108] Take a 96-well spotting plate, add 6 μl of sample buffer to eac...

Embodiment 3

[0119] [Example 3] Direct Sequencing of PCR Amplified Product of Purified OTOF Gene Coding Region

[0120] 1. Purity and dosage requirements of PCR product DNA template

[0121] DNA purity: OD 260 / OD 280 =1.6~2.0.

[0122] DNA concentration: PCR product 10ng / μl.

[0123] DNA consumption:

[0124] PCR product

[0125]

[0126] 2. Sequencing reaction

[0127] 1. The reagents required for the sequencing reaction should be freshly prepared, and the reagents that need to be sterilized by autoclaving must be sterilized before use. The equipment required for the sequencing reaction (such as 384-well plates, tips, etc.) should also be clean and sterile.

[0128] 2. In order to ensure the freshness of sequencing samples and reaction reagents, it should be operated on ice when adding samples.

[0129] 3. The current reaction system is 5 μl, and the amount of various reagents added is shown in Table 2.

[0130] Table 2 Sequencing reaction system of OTOF gene PCR amplificat...

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Abstract

The present invention provides a kit for detecting sensorineural deafness and auditory neuropathy or auditory neuropathy spectrum disorders (ANSD) associated c.1671_1673del G (X579) mutation on an OTOF gene. The kit comprises a reagent for extracting DNA from a sample to be tested, a PCR reagent for amplification of the sample DNA, and a reagent for sequencing of the products from the PCR amplification. The PCR reagent for amplification of the DNA comprises a PCR primer; and target fragments amplified by the PCR primer comprise 1671st-1673rd base of the OTOF gene. The kit can be used to detect whether a patient has the mutation gene, so as to diagnose causes and types of sensorineural deafness and auditory neuropathy or ANSD. The mutation gene and the detection method are beneficial to clinical implement of OTOF mutation screening on patients with sensorineural deafness and auditory neuropathy or ANSD, and provide basis for diagnosis and treatment of patients with sensorineural deafness and auditory neuropathy or ANSD.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a kit for detecting OTOF gene mutation; the invention also relates to a new OTOF mutation gene and its use in the diagnosis and / or treatment of sensorineural deafness and auditory neuropathy / auditory neuropathy spectrum disorder application. Background technique [0002] Auditory Neuropathy Spectrum Disorders (ANSD) is a special sensorineural deafness caused by damage to the auditory branch of the cranial nerve VIII. It is an abnormal auditory function disease that manifests as sound can enter the inner ear normally through the outer ear and middle ear, but the sound signal cannot be transmitted from the inner ear to the brain synchronously. Clinical hearing examination shows a group of auditory dysfunction syndrome with normal evoked otoacoustic emissions and severe abnormal auditory brainstem response. Auditory neuropathy spectrum disorders usually start in childhood or adolesce...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 王秋菊
Owner 王秋菊
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