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hpr1 gene mutant and its application in the preparation of deafness diagnostic reagents

A technology of diagnostic reagents and detection reagents, which is applied in the fields of application, genetic engineering, plant gene improvement, etc., and can solve the problems of delayed disease, aggravated disease, difficult detection and diagnosis of hearing loss, etc.

Active Publication Date: 2022-06-07
NANTONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The occurrence of many deafnesses is accompanied by the process of gradual hearing loss. Early hearing loss is not easy to be found and diagnosed, which leads to delay and aggravation of the disease
However, there is no research report on the relationship between HPR1 and hearing loss and deafness

Method used

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  • hpr1 gene mutant and its application in the preparation of deafness diagnostic reagents
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  • hpr1 gene mutant and its application in the preparation of deafness diagnostic reagents

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1. Verification of the correlation between HPR1 and deafness diseases

[0051] The first step, sample preparation: clinically discover a family of acquired deafness (such as figure 1 Among them, 15 patients with acquired deafness have been diagnosed, and the peripheral blood samples of the deaf patients in the family (experimental group, 9 people) and non-deaf individuals (18 people) were extracted respectively, and the total RNA of each sample was extracted by TRIzol method, and the Store at -80°C until use.

[0052] The second step, sample exome sequencing and pathogenic mutation gene analysis: use whole exome sequencing technology to sequence and analyze each RNA sample. The specific contents include: using the Illumina TruSeq Exome Enrichment Kit to capture the sequences of exons and surrounding introns of 20,794 expressed genes contained in each sample, and to determine the sequences of microRNA and other non-coding genes. The measurement instrument is HiS...

Embodiment 2

[0055] Example 2 Preparation of the kit of the present invention

[0056] The sequence of the HPR1 mutant is shown in SEQ ID: NO: 2. Its specific PCR upstream and downstream primers were designed by Primer 5, and the primers were synthesized by Invitrogen. The purity was PAGE grade. The synthesized primers were dissolved in RNase free H2O. The concentration is 10 μM.

[0057] Prepare a kit that includes the following components:

[0058] (a) Extraction system:

[0059] 1) Trizol reagent, 2 tubes, 2000μL / tube;

[0060] 2) Chloroform, 1 tube, 500 μL / tube;

[0061] 3) Absolute ethanol, 1 tube, 8000μL / tube;

[0062] 4) RNase free ddH 2 O, 2 tubes, 2000μL / tube;

[0063] 5) Isopropanol, 8000μL / tube;

[0064] (b) Reverse transcription system:

[0065] 1) Total RNA reverse transcription primer Oligo dT, 1 tube, concentration: 50 μM, 50 μL / tube;

[0066] 2) Reverse transcriptase (200U / μL) 50μL;

[0067] 3) 50μL of dNTP Mixture (10mM each);

[0068] 4) 50μL of reverse transcr...

Embodiment 3

[0076] Example 3 Sequence detection of HPR1 gene in peripheral blood cells of deaf patients

[0077] After adding TRIzol to the peripheral blood cells, it was left at room temperature for 10 min to fully lyse the sample. Add 200 μl of chloroform to each 1 ml of TRIzol, shake vigorously and mix, and leave at room temperature for 3-5 minutes to allow natural phase separation. Centrifuge at 12,000 rpm for 15 min at 4°C. The sample will separate into three layers: the yellow organic phase, the middle layer, and the colorless aqueous phase. The RNA is mainly in the aqueous phase. Transfer the aqueous phase to a new tube. An equal volume of ice-cold isopropanol was added to the supernatant and left at room temperature for 15 min. Centrifuge at 12,000 rpm at 4°C for 10 min, discard the supernatant, and precipitate the RNA at the bottom of the tube. Add 1 ml of 75% ethanol to the RNA pellet, and gently shake the centrifuge tube to suspend the pellet. Add 1 ml of 75% ethanol per 1 ...

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Abstract

The invention discloses a gene mutant of HPR1 and its relationship with autosomal dominant non-syndromic deafness. The mutation site related to deafness is the 547th nucleotide C mutation in the coding region of HPR1 gene to G, It leads to the mutation of amino acid 183 from L to V after translation (c.547C>G, p.L183V). The invention discloses the application of HPR1 gene mutant in the preparation of deafness diagnostic reagents and a diagnostic kit for deafness detection. In the present invention, through exome sequencing analysis and animal experiment verification on deafness family members, it is found that HPR1 has a significant correlation with the occurrence of deafness, and is an important inducement for the occurrence of hereditary deafness, and can be used for early diagnosis of clinical deafness patients. And provide a theoretical basis for early intervention and treatment of deafness.

Description

technical field [0001] The invention relates to the field of disease-related mutant genes and molecular diagnosis in the field of biomedicine, in particular to a mutation site of HPR1 gene related to autosomal dominant non-syndromic deafness in a family with delayed-onset deafness. HPR1 is used in the preparation of deafness diagnostic reagents. The application in HPR1 and a HPR1 diagnostic kit for deafness can diagnose deafness and hearing loss-related diseases according to the nucleic acid sequence characteristics of individual HPR1. Background technique [0002] Deafness is one of the most serious diseases of human beings. Patients often lose the ability of normal language communication due to deafness, and suffer great pain and inconvenience in the spirit and life. The incidence of neonatal deafness in the world is about 1‰, about 15% of adults have some degree of hearing loss, and 35-55% of people over 60 years old have different degrees of hearing impairment. At prese...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12N15/12
Inventor 刘东段旭初张鲁平
Owner NANTONG UNIVERSITY
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