HPR1 gene mutant and application thereof for preparing diagnostic reagent for deafness

A technology of diagnostic reagents and diagnostic kits, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as aggravation, delayed disease, and lack of HPR1.

Active Publication Date: 2019-06-07
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, w

Method used

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  • HPR1 gene mutant and application thereof for preparing diagnostic reagent for deafness
  • HPR1 gene mutant and application thereof for preparing diagnostic reagent for deafness
  • HPR1 gene mutant and application thereof for preparing diagnostic reagent for deafness

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 found a family of acquired deafness (such as figure 1 shown), in which 15 patients with acquired deafness have been diagnosed, the peripheral blood samples of deaf patients (experimental group, 9 people) and non-deaf individuals (18 people) in the family were extracted respectively, and the total RNA of each sample was extracted by TRIzol method, and Store at -80°C until use.

[0052] The second step, sample exome sequencing and disease-causing mutation gene analysis: use the whole exome sequencing technology to sequence and analyze each RNA sample. The specific content includes: use the Illumina TruSeq Exome Enrichment Kit to capture the sequences of exons and surrounding intron regions of 20,794 expressed genes contained in each sample, and measure the sequences of microRNA sequences and other non-coding genes. The measuring instrument ...

Embodiment 2

[0055] Embodiment 2 prepares kit of the present invention

[0056] The sequence of the HPR1 mutant is shown in SEQ ID: NO: 2, and its specific PCR upstream and downstream primers are designed by Primer 5, and Invitrogen Company is responsible for primer synthesis, with a purity of PAGE grade. The synthesized primers are dissolved in RNase free H2O, and The concentration is 10 μM.

[0057] Prepare a kit comprising 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, 8000 μL / tube;

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

[0063] 5) Isopropanol, 8000 μL / 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) dNTP Mixture (10mM each) 50μL;

[0068] 4) Reverse trans...

Embodiment 3

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

[0077] After adding TRIzol to peripheral blood cells, place it at room temperature for 10 minutes to fully lyse the samples. Add 200 μl of chloroform to every 1ml of TRIzol, vibrate vigorously and mix well, then place at room temperature for 3-5min to allow natural phase separation. Centrifuge at 12,000 rpm at 4°C for 15 min. The sample will separate into three layers: a yellow organic phase, an intermediate layer and a 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 1ml of 75% ethanol to the RNA pellet, shake the centrifuge tube gently, and suspend the pellet. Add 1 ml of 75% ethanol per 1 ml...

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Abstract

The invention discloses an HPR1 gene mutant, as well as relationship between the HPR1 gene mutant and autosomal dominant nonsyndromic deafness. The deafness-related mutation site is the nucleotide 547of the HPR1 gene coding region at which C mutates to G; and the mutation causes the amino acid 183 to mutate, after translation, from L to V (c. 547C > G, p. L183V). The invention discloses application of the HPR1 gene mutant for preparing a diagnostic reagent for deafness, as well as a diagnostic kit for deafness. By performing exome sequencing and animal experiment validation on family memberswith deafness, the invention discovers significant correlation between HPR1 and deafness, namely, the HPR1 is an important inducement of hereditary deafness; and thus, the HPR1 can be used for early clinical diagnosis of patients with deafness. Moreover, theoretical basis can be provided for early intervention and treatment on the deafness.

Description

technical field [0001] The invention relates to the field of disease-related mutation genes and molecular diagnosis in the field of biomedicine, in particular to the mutation site of the HPR1 gene related to autosomal dominant non-syndromic deafness in a family of hereditary delayed-onset deafness. HPR1 is used in the preparation of deafness diagnostic reagents application in , and a deafness HPR1 diagnostic kit, which 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 human diseases. Patients often lose the ability to communicate normally due to deafness, and suffer great pain and inconvenience in terms of spirit and life. The incidence of newborn 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 present,...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/12
Inventor 刘东段旭初张鲁平
Owner NANTONG UNIVERSITY
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