Fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for mutation A1555G of mitochondrial deafness and application of fluorescent quantitative PCR detection kit

A detection kit and fluorescence quantitative technology are applied in the fields of molecular biology and genetic engineering, which can solve the problems of incomplete fluorescence quenching and achieve the effect of simple diagnosis.

Inactive Publication Date: 2017-10-24
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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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 (Polymerase Chain Reaction) detection kit for mutation A1555G of mitochondrial deafness and application of fluorescent quantitative PCR detection kit
  • Fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for mutation A1555G of mitochondrial deafness and application of fluorescent quantitative PCR detection kit
  • Fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for mutation A1555G of mitochondrial deafness and application of fluorescent quantitative PCR detection kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] see figure 1 , the fluorescent quantitative PCR detection kit of the mitochondrial deafness A1555G mutation provided by the present invention is composed of quantitative PCR reaction solution (1), the first positive control substance (2), the second positive control substance (3), and the negative control substance (4) , instructions (5) and box body (6), wherein the quantitative PCR reaction solution contains PCR buffer, MgCl 2 , dNTPs, thermostable DNA polymerase, upstream amplification primer, downstream amplification primer, first fluorescent probe and second fluorescent probe.

[0026] The upstream amplification primer sequence is: 5'-CATTTAACTAAAACCCCTACGCATTT-3'

[0027] The downstream amplification primer sequence is: 5'-GCACTTTCCAGTACACTTACCATGTT-3'

[0028] The first fluorescent probe sequence is: 5'-FAM-AGGAGGCAAGTCG-MGB-3'

[0029] The sequence of the second fluorescent probe is: 5'-HEX-TAGAGGAGACAAGTCG-MGB-3'

[0030] The first positive control substanc...

Embodiment 2

[0032] Example 2 Application of Mitochondrial Deafness A1555G Mutation Fluorescent Quantitative PCR Detection Kit

[0033] (1) Test samples:

[0034] Thirty children with sensorineural deafness were diagnosed in Children's Hospital Affiliated to Zhejiang University School of Medicine. Mitochondrial DNA was extracted from peripheral blood samples in all cases as test samples using the mitochondrial DNA extraction kit from BioVision, USA.

[0035] (2) Fluorescent quantitative PCR detection

[0036] Add 5 μl of the test sample, positive control substance or negative control substance to the quantitative PCR reaction solution tube, and perform PCR amplification on a two-color (or above) fluorescent quantitative PCR instrument. The FAM and HEX channels are used to detect the G at site 1555 respectively base and A base. The PCR reaction conditions were pre-denaturation at 94°C for 5 minutes, 20 seconds at 94°C → 60 seconds at 60°C, a total of 40 cycles.

[0037] (3) Test results

...

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Abstract

The invention provides a fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for common mutation A1555G of mitochondrial deafness. The kit comprises quantitative PCR reaction solutions, a first positive control, a second positive control, a negative control, a description and a box body, wherein the quantitative PCR reaction solutions contain a PCR buffer solution, MgCl2, dNTPs, 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 A>G mono-base mutation of a 1555th locus of mtDNA, and whether the locus is subjected to A>G mutation or not is determined through detecting 1555th nucleotide of a mitochondrion correlated to deafness by fluorescent quantitative PCR, so that the kit has the advantages of simplicity, convenience, rapidness, accuracy and the like, can be applied to the clinical diagnosis and control of deafness related to mitochondrial mutation A1555G and can also be used for providing a foundation for preventing and treating the mitochondrial deafness induced by the mutation A1555G.

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 A1555G mutation and its application in the detection of mitochondrial deafness A1555G mutation. 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] The A1555G mutation located on mtDNA 12S rRNA ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2531/113C12Q2561/101C12Q2545/113
Inventor 舒强尚世强李伟陶然
Owner ZHEJIANG UNIV
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