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Kit for detecting deafness susceptibility genes

A susceptibility gene and kit technology, applied in the field of detection of deafness susceptibility genes, can solve the problems of indeterminacy, time-consuming and laborious, and achieve the effects of fast detection turnaround time, reduced sample manipulation, and high sensitivity

Inactive Publication Date: 2017-02-01
DALIAN GENTALKER BIO-TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These traditional methods are not qualitative, or time-consuming and labor-intensive. More importantly, these methods are difficult to detect multiple mutation sites of different genes at the same time.

Method used

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  • Kit for detecting deafness susceptibility genes
  • Kit for detecting deafness susceptibility genes
  • Kit for detecting deafness susceptibility genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] The design of embodiment 1 primer

[0054] (1) PCR amplification primers:

[0055] According to the selected deafness susceptibility genes, the amplification primers specific for each deafness susceptibility gene are designed, and the amplification primers can amplify a DNA sequence including the mutation site. The amplification primer has at least 15 bases at the 3' end that completely match the gene sequence it is targeting, and has a 10 base (acgttggatg) tag sequence at the 5' end to distinguish the amplification primer from the extension primer.

[0056] (2) Mass spectrometry extension primer:

[0057] Design an extension primer, the length of the extension primer is 15-28 bases, and its 3' end is located at the previous base of the relevant mutation site, and the extension primer only extends one base when the extension reaction occurs, and the extended The base is the relevant mutation site.

[0058] The information on the mutation site of the deafness suscepti...

Embodiment 2

[0069] sample preparation

[0070] Sampling method: Use a cotton swab to wipe the inside of the cheek 15 times.

[0071] DNA was extracted (extracted using Tiangen DNA kit, and the operation method was extracted according to the instructions of the kit).

Embodiment 3

[0072] The detection method of embodiment 3 deaf susceptibility genes

[0073] (1) Using the DNA extracted in Example 2 as a template, use the PCR amplification primers in Example 1 to amplify by PCR to obtain the target sequence amplification product. See Table 4 for the PCR amplification reaction system. Among them, all reagents were purchased from Agena Bioscience.

[0074] Table 4: PCR amplification reaction system

[0075]

[0076] The PCR reaction conditions were 94°C for 2 minutes; denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 1 minute, and a total of 45 cycles of amplification; the final extension at 72°C for 5 minutes. The DNA extracted in 2 was used as the DNA template for PCR amplification. At the same time, sterile double distilled water was used as a negative control. The control sample and the test sample were reacted and tested according to the same reaction process to verify the validity of the test.

[007...

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Abstract

The invention discloses a kit for detecting deafness susceptibility genes, namely the invention provides a kit for rapidly and accurately detecting the GJB2, SLC26A4 and 12SrRNA deafness susceptibility genes by virtue of an MALDI-TOF mass-spectrometry technique. The kit mainly consists of the following ingredients: PCR amplification products shown as SEQ ID NO:1-SEQ ID NO:32 and mass-spectrometry extension primers shown as SEQ ID NO:33-SEQ ID NO:48. The kit provided by the invention, by combining the mass-spectrometry technique and a multi-primer extension technique, is relatively simple in adopted reagent consumables, low in cost and stable, and dozens to thousands of samples are analyzed in one reaction, so that loss caused by reagent preparation is reduced; and the kit is quite high in sensitivity, accuracy and detection consistency, and detection throughput is improved.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a method and a kit for detecting deafness susceptibility genes by combining multiplex PCR amplification technology and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Background technique [0002] Deafness is a common congenital disease that seriously affects the quality of human life. It can be caused by a single gene mutation or a compound mutation of different genes, or by environmental factors (such as drugs) or a combination of both genes and the environment. Studies have shown that hereditary deafness has a high degree of genetic heterogeneity. Gene diagnosis is of great significance to the clinical evaluation of deaf patients and their families. The identification of deafness responsible genes is the molecular diagnosis of patients, the detection of gene carriers and prenatal diagnosis. One of the prerequisites for diagnosis. It i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q1/6883C12Q2600/16C12Q2531/113C12Q2537/143C12Q2565/627
Inventor 董新波刘琦赵金银方楠于闯许立志李杰
Owner DALIAN GENTALKER BIO-TECH CO LTD
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