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Primer probe combination for deafness gene mutation detection and application thereof

A deafness gene and primer-probe technology, applied in the field of molecular biology, can solve the problem that primers and probes cannot be accurately detected, and achieve the effects of eliminating nucleic acid cross-contamination, high reagent stability and high specificity

Inactive Publication Date: 2018-11-16
WUXI SHENRUI BIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, not only the probes designed according to the relevant sites can be detected, but there are also situations where the primers and probes designed according to the relevant sites cannot be accurately detected.

Method used

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  • Primer probe combination for deafness gene mutation detection and application thereof
  • Primer probe combination for deafness gene mutation detection and application thereof
  • Primer probe combination for deafness gene mutation detection and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] The assembly of embodiment 1 kit

[0087] The design of the primer probe combination, the specific sequence is shown in Table 1 below:

[0088] Table 1

[0089]

[0090] Negative quality control: the negative quality control is TE buffer;

[0091] Positive quality control: deafness gene mutation plasmid;

[0092]Auxiliary reagents: Taq enzyme, dNTPs, MgCl 2 and PCR reaction buffer;

[0093] The above components, instructions and centrifuge tubes are assembled and packed into the kit.

Embodiment 2

[0094] Example 2 Assembly of a device for detecting deafness gene mutations

[0095] This embodiment provides a device for detecting deafness gene mutations, including:

[0096] (1) Extraction unit: used to extract sample DNA;

[0097] (2) Preparation reaction unit: connected to the extraction unit, used to prepare the reaction system;

[0098] Wherein, the reaction system includes PCR buffer, Taq enzyme, dNTPs, MgCl 2 , Deafness gene mutation detection-specific primer pair, deafness gene-specific probe, amplification blocking probe, internal reference primer pair, internal reference probe, positive quality control and negative quality control;

[0099] The MgCl 2 The concentration of the dNTPs is 1-8mM, the concentration of the dNTPs is 0.1-1mM, the concentration of the Taq enzyme is 0.02-0.08U / μl, the concentration of the deafness gene mutation detection specific primer pair is 500-900nM, the The concentration of the deafness gene-specific molecular beacon probe is 100-1...

Embodiment 3

[0102] Example 3 Deafness Gene Mutation Detection

[0103] Using the kit in Example 1 and the device in Example 2 to detect the mutation of the deafness gene comprises the following steps:

[0104] (1) Sample DNA extraction:

[0105] The applicable sample is fingertip capillary whole blood. Use a column blood DNA extraction kit or other appropriate extraction methods. The concentration and purity of the extracted DNA need to be measured with a UV spectrophotometer. The DNA OD260 / OD280 value should be 1.7 to 1.9 , the concentration should be between 0.5 and 20ng / μl (if the concentration is lower than 0.5ng / μl, it is recommended to use alcohol precipitation to concentrate the extracted DNA to make the concentration meet the requirements; if the concentration is higher than 20ng / ul, it should be properly diluted to specified concentration range);

[0106] (2) preparation system, the concrete system composition is as shown in table 2 below:

[0107] Table 2

[0108]

[0109...

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Abstract

The invention relates to a detection production of gene mutation and in particular to a primer probe combination for deafness gene mutation detection. The primer probe combination comprises a detection primer probe. The detection primer probe comprises a deafness gene mutation detection specific primer pair and a deafness gene specific molecular beacon probe. Sites of the mutation detection for adeafness gene are IVS7-2, GJB2, 12SrRNA-1494 and 12SrRNA-1555 mutation sites. The primer probe combination has the advantages of strong specificity, high sensitivity, simple and rapid operation to thedeafness gene mutation, a detection result has the better accuracy and repeatability, is capable of assisting clinic therapy, and has an important value.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a product for detecting gene mutations, in particular to a combination of primers and probes for detecting deafness gene mutations and its application. Background technique [0002] Deafness is one of the most common genetic diseases clinically, and it is also a common disease affecting human health. According to statistics, there are 27.8 million people with hearing and speech disabilities in China, accounting for 34% of the total number of disabled people in the country. Among them, there are 20.04 million people with simple hearing disabilities, and the annual growth rate is 20,000 to 30,000 newborn deaf children; Beijing In the newborn deafness gene screening conducted by the Health Bureau this year, nearly 9,000 of the nearly 200,000 newborns in the city were found to be positive for common deafness genes, with a positive rate of 4.55%. The prevalence of deafness in...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6883C12Q2600/156C12Q2563/107C12Q2545/114
Inventor 白静盛青松
Owner WUXI SHENRUI BIO PHARMA
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