Kit for detecting 2975-2978delAG mutation of OTOF gene

A technology for detecting reagents and kits, which is applied in the field of kits for detecting OTOF gene 2975-2978delAG mutations, to achieve the effect of avoiding economic losses

Inactive Publication Date: 2009-11-25
GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In patients with TYPE I auditory neuropathy, the lesion is deep in

Method used

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  • Kit for detecting 2975-2978delAG mutation of OTOF gene
  • Kit for detecting 2975-2978delAG mutation of OTOF gene
  • Kit for detecting 2975-2978delAG mutation of OTOF gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] 1. Preparation of blood sample DNA of the subject to be tested

[0050] 1. Research object

[0051] 76 cases of sporadic non-syndromic auditory neuropathy patients and 92 normal hearing controls without family history were screened for OTOF gene according to the following method. Among the 76 patients, 1 patient not only had the abnormal auditory function characteristics of auditory neuropathy, but also showed speech recognition, discrimination and hearing threshold changes when the body temperature changed slightly. After functional examination, a diagnosis of temperature-sensitive auditory neuropathy was made. OTOF gene detection in this patient with temperature-sensitive auditory neuropathy revealed that the patient was a compound heterozygote for the 2975-2978delAG mutation and another mutation. The mother of the patient was a carrier of the simple 2975-2978delAG mutation, and the father was a carrier of another mutation. The 2975-2978delAG mutation was not found...

Embodiment 2

[0084] 1. Purification of PCR products——96-well plate method

[0085] 1. Add 50 μl sterile water to the 96-well plate containing the PCR product and mix well.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 5. Store in a -20°C refrigerator.

[0090] 2. Quantification by electrophoresis

[0091] 1. Sample preparation

[0092]Take a 96-well spotting plate, add 6 μl of sample buffer to each well, remove the PCR product (2 μl) from each well of the cavity plate containing the PCR product, transfer to...

Embodiment 3

[0105] 1. Purity and dosage requirements of PCR product DNA template

[0106] DNA purity: OD 260 / OD 280 = 1.6 to 2.0.

[0107] DNA concentration: PCR product 10ng / μl.

[0108] DNA consumption:

[0109] PCR product

[0110] 100-200bp 1-3ng

[0111] 200-500bp 3-10ng

[0112] 500-1000bp 5-20ng

[0113] 1000-2000bp 10-40ng

[0114] >2000bp 40-100ng

[0115] 2. Sequencing reaction

[0116] 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.

[0117] 2. In order to ensure the freshness of sequencing samples and reaction reagents, it should be operated on ice when adding samples.

[0118] 3. The current reaction system is 5 μl, and the amount of various reagents added is shown in Table 2.

[0119] The sequencing reaction system of t...

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Abstract

The present invention relates to an OTOF mutant gene with 2975-2978delAG mutation, wherein the mutant gene is related with the auditory neuropathy. The invention provides a kit for detecting 2975-2978delAG mutation of OTOF gene, and a detecting method for detecting the cause and the type of the auditory neuropathy through detecting whether the mutant gene exists in the patient. The mutant gene and detecting method of the invention facilitate the clinically development of OTOF mutation screening operation of the auditory neuropathy patient, and provide criterion for diagnosing and treating the neuropath.

Description

technical field [0001] The invention relates to a kit for detecting the 2975-2978delAG (X1000) mutation gene of the OTOF gene. [0002] The invention also relates to a new OTOF mutant gene and its application in diagnosing and / or treating auditory neuropathy. Background technique [0003] Auditory neuropathy (AN) is a special sensorineural deafness caused by damage to the auditory branch of the VIII cranial nerve. 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 usually starts in childhood or adolescence, with an incidence of about 0.23% among high-risk infants and as high as 11% among deaf children with ABR abnor...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 王秋菊赵亚丽王大勇
Owner GENERAL HOSPITAL OF PLA
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