Fluorescent quantitative PCR detection kit for mitochondrion deafness A7445G mutation and application thereof

A detection kit and fluorescence quantitative technology, applied in the fields of molecular biology and genetic engineering, can solve the problem of incomplete fluorescence quenching

Pending Publication Date: 2017-10-20
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the problem of incomplete fluorescence quenching of traditional Taqman probes for fluorescent quantitative PCR, ABI Company of the United States introduced a new Taqman probe——MGB pro

Method used

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  • Fluorescent quantitative PCR detection kit for mitochondrion deafness A7445G mutation and application thereof
  • Fluorescent quantitative PCR detection kit for mitochondrion deafness A7445G mutation and application thereof
  • Fluorescent quantitative PCR detection kit for mitochondrion deafness A7445G mutation and application thereof

Examples

Experimental program
Comparison scheme
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Example Embodiment

[0024] Example 1

[0025] See figure 1 , The fluorescent quantitative PCR detection kit for mitochondrial deafness A7445G mutation provided by the present invention consists of a quantitative PCR reaction solution (1), a first positive control substance (2), a second positive control substance (3), and a negative control substance (4) , Instructions (5) and box (6), where the quantitative PCR reaction solution contains PCR buffer, MgCl 2 , DNTPs, heat-resistant DNA polymerase, upstream amplification primer, downstream amplification primer, first fluorescent probe and second fluorescent probe.

[0026] The sequence of the upstream amplification primer is: 5’-CACCCTACCACACATTCGAAGA-3’

[0027] The downstream amplification primer sequence is: 5’-TGGCTTGAAACCAGCTTTGG-3’

[0028] The first fluorescent probe sequence is: 5’-FAM-CCGTATACATAAAATCTAGGCA-MGB-3’

[0029] The second fluorescent probe sequence is: 5’-HEX--MGB-3’

[0030] The first positive control is a DNA sample with the A base at...

Example Embodiment

[0032] Example 2 Application of mitochondrial deafness A7445G mutation fluorescence quantitative PCR detection kit

[0033] (1) Test samples:

[0034] In the Children's Hospital of Zhejiang University School of Medicine, 35 cases of children with sensorineural hearing loss were diagnosed. All cases used the mitochondrial DNA extraction kit from BioVision of the United States to extract mitochondrial DNA from peripheral blood samples as test samples.

[0035] (2) Fluorescence quantitative PCR detection

[0036] Add 5μl of test sample, positive control substance or negative control substance into the quantitative PCR reaction solution tube, and perform PCR amplification on a two-color (or above) fluorescent quantitative PCR instrument. FAM and HEX channels are used to detect G at 7445 site respectively. Bases and A bases. The PCR reaction conditions were 94°C for 5 minutes pre-denaturation, 94°C for 20 seconds→60°C for 60 seconds, a total of 40 cycles.

[0037] (3) Test results

[0038] ...

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Abstract

The invention provides a fluorescent quantitative PCR detection kit for mitochondrion deafness A7445G mutation. The kit is prepared from quantitative PCR reaction liquid, a first positive control substance, a second positive control substance, a negative control substance, a specification and a box body, wherein the quantitative PCR reaction liquid is prepared from a PCR buffer solution, MgCl2, dNTPs, a heat-resistant DNA polymerase, an upstream amplification primer, a downstream amplification primer, a first fluorescent probe and a second fluorescent probe. According to the kit provided by the invention, two MGB probes are designed for the A being larger than G single-base mutation on the site 7445 of mtDNA; the nucleotide on the site 7445 of the deafness related mitochondrion is detected through fluorescent quantitative PCR to determine whether the A being larger than G mutation happens on the site; the kit is simple, quick, accurate and the like and can be applied to clinical diagnosis and control of mitochondrion A7445G mutation related deafness and also provides a basis for the prevention and treatment of mitochondrion deafness caused by A7445G mutation.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and genetic engineering, and specifically relates to a fluorescent quantitative PCR detection kit for mitochondrial deafness A7445G mutation and its application. Background technique [0002] Deafness is a public health problem of global concern, which is caused by genetic and environmental factors, and more than 50% of deafness patients are caused by genetic factors. In hereditary deafness, 30% are syndromic deafness and 70% are non-syndromic deafness. The inheritance mode of deafness can show autosomal dominant inheritance, autosomal recessive inheritance, X-linked inheritance and maternal inheritance. Mitochondrial DNA (mtDNA) mutation is one of the important causes of maternal hereditary deafness, and the incidence of hereditary deafness is The rate is about 1% to 2%. [0003] Deafness-associated mtDNA tRNA gene mutations involve a total of 16 tRNAs, among which tRNA Ser(UCN) Gen...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2545/101C12Q2563/107C12Q2561/101
Inventor 舒强尚世强李伟陶然
Owner ZHEJIANG UNIV
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