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Cpf1 kit for rapid detection of hereditary deafness pathogenic gene SLC26A4 mutation and detection method thereof

A technology of hereditary deafness and detection method, which is applied in the field of rapid detection method and detection kit for multiple mutation sites of SLC26A4 gene, can solve the problems of difficult data analysis, high sequencing cost, long detection time, etc., and achieves convenience and speed. The effect of result interpretation, high specificity and rapid visual detection

Active Publication Date: 2020-03-13
国家卫生健康委科学技术研究所
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are many detection technologies for deafness, mainly including allele-specific PCR, microarray gene chips, nucleic acid mass spectrometry and sequencing technologies, etc. These technologies either have high detection throughput or accurate determination of height, but the disadvantages are The main source of test samples is adult venous blood or neonatal heel blood, which is invasive sampling, and the sample preparation process is cumbersome, the detection time is long, the sequencing cost is high, and data analysis is difficult. The sensitivity developed based on these technologies is high. Highly specific, fast and convenient non-invasive testing

Method used

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  • Cpf1 kit for rapid detection of hereditary deafness pathogenic gene SLC26A4 mutation and detection method thereof
  • Cpf1 kit for rapid detection of hereditary deafness pathogenic gene SLC26A4 mutation and detection method thereof
  • Cpf1 kit for rapid detection of hereditary deafness pathogenic gene SLC26A4 mutation and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Rapid and sensitive detection of SLC26A4 gene mutation site fragments

[0042] 1.1 Nucleic acid preparation

[0043] In this case, the SLC26A2 gene fragment is based on the SLC26A4 gene sequence in the NCBI database. Amplification primers are designed according to each mutation site, and human genomic DNA is used as a template. After PCR amplification to obtain the target fragment, it is constructed by homologous recombination On the pUC57 vector, named respectively as pUC57-IVS7-2-A (SEQ NO.1), pUC57-IVS7-2-G (SEQ NO.2), pUC57-1174-A (SEQ NO.3), pUC57-1174 -T(SEQ NO.4), pUC57-1229-C(SEQ NO.5), pUC57-1229-T(SEQ NO.6), pUC57-2168-A(SEQ NO.7), pUC57-2168-> G (SEQ NO. 8). The amplification system is shown in Table 1.

[0044] Table 1. SLC26A4 Gene Mutation Site Fragment Amplification System

[0045]

[0046] The reaction system is as follows:

[0047]

[0048] After the sample is obtained, the next step of nucleic acid detection is carried out.

[00...

Embodiment 2

[0064] Example 2: Optimization of conditions for rapid detection of SLC26A4 gene fragments

[0065] The rapid detection of SLC26A4 gene mutation site is helpful to clearly judge the mutation in clinical samples, which is of great significance to the detection of early deafness. In this implementation case, taking the IVS7-2A>G, c.2168A>G, c.1229C>T and c.1174A>T sites of the SLC26A4 gene as an example, the Cpf1 detection kit of the present invention is used to demonstrate the pathogenicity of SLC26A4 Optimization of the detection conditions of the site.

[0066] Based on the fact that Cpf1 performs highly specific recognition and cleavage of the target sequence under the guidance of crRNA, a hypothesis is proposed: the shortening of the length of crRNA precisely paired with the SLC26A4 gene can lead to the inability of the Cpf1 cleavage system to efficiently recognize, which is manifested in the detection of fluorescence values ​​at a certain level. The degree is reduced but ...

Embodiment 3

[0077] Example 3: Rapid and sensitive typing of a pair of SLC26A4 gene mutation sites using the protocol

[0078] Vestibular aqueduct enlargement / Pendred syndrome is an autosomal recessive genetic disease, and the typing detection of the mutation site of the causative gene SLC26A4 is helpful to clearly determine whether the clinical samples are normal people, mutation carriers or deaf patients. The detection of early deafness is of great significance. In this implementation case, taking the IVS7-2A>G site of the SLC26A4 gene as an example, the Cpf1 detection kit of the present invention is used to type the deafness pathogenic site.

[0079] Based on the fact that Cpf1 affects the cutting efficiency according to the degree of matching between crRNA and the target sequence, a hypothesis is put forward: select a suitable control sequence near the IVS7-2A>G site, transcribe it into IVS7-2-G-ctrl in vitro, and convert IVS7-2-G The detection signal of IVS7-2-G-ctrl is compared with...

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Abstract

The invention discloses a Cpf1 kit for rapid detection of hereditary deafness pathogenic gene SLC26A4 mutation and a detection method thereof. The Cpf1 kit comprises a Cpf1 detection system. The Cpf1detection system comprises: a specific crRNA for SLC26A4, a Cpf1 protein and a single-stranded DNA reporting system. The specific crRNA is any one or more designed and synthesized for mutation sites.The single-stranded DNA reporting system comprises an ssDNA FQ reporter for fluorescence detection by an enzyme-labeled instrument and / or an ssDNA DB reporter for immune colloidal gold strip detection. By detecting SLC26A4 gene mutation sites with the Cpf1 for the first time, the kit of the invention has the advantages of high sensitivity, strong specificity, low time consumption, no dependence onlarge-scale experimental equipment and the like.

Description

technical field [0001] The invention relates to the field of genetic detection of hereditary deafness, in particular to a rapid detection method and detection kit for multiple mutation sites of the SLC26A4 gene used in the clinical diagnosis of vestibular aqueduct enlargement / Pendred syndrome, belonging to the field of biotechnology . Background technique [0002] Deafness is a common disease of the auditory nervous system in clinical practice, which seriously affects the quality of life of human beings. There are many reasons for deafness, including genetic factors, environmental factors, drugs, trauma, infection, etc., which can all lead to deafness. The global incidence of newborn deafness is about 1 / 1000, of which more than 50% is caused by genetic factors. In hereditary deafness, about 80% are autosomal recessive inheritance. Although deafness genes have high gene and locus heterogeneity, most hereditary deafness is caused by mutations in a few hotspot genes, including...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2531/119C12Q2563/107C12Q2525/204C12Q2565/625Y02A50/30
Inventor 马旭王鑫杰金敏张璐金孝华
Owner 国家卫生健康委科学技术研究所
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