Fluorescent quantitative PCR detection kit for mitochondrial deafness C1494T mutation and application thereof
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A detection kit and fluorescence quantitative technology, applied in the fields of molecular biology and genetic engineering, can solve the problems of incomplete fluorescence quenching
Inactive Publication Date: 2017-11-24
ZHEJIANG UNIV
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[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 probe in 2000. Its 3' end uses a non-fluorescent quenching group. It does not emit light after absorbing the energy of the reporter group, which greatly reduces the interference of the background signal
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Embodiment 1
[0025] see figure 1 , the fluorescent quantitative PCR detection kit for the mitochondrial deafness C1494T mutation provided by the present invention is composed of a quantitative PCR reaction solution 1, a first positive control substance 2, a second positive control substance 3, a negative control substance 4, an instruction manual 5 and a 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'-GCCCTGAAGCGCGTACAC-3'
[0027] The downstream amplification primer sequence is: 5'-CCATGTTACGACTTGTCTCCTCTATATAA-3'
[0028] The first fluorescent probe sequence is: 5'-FAM-CCGTCACTTCTCCT-MGB-3'
[0029] The second fluorescent probe sequence is: 5'-HEX-CCGTCACCCTCCT-MGB-3'
[0030] The first positive control substance is a DNA sample whos...
Embodiment 2
[0032] Example 2 Application of Fluorescent Quantitative PCR Detection Kit for Mitochondrial Deafness C1494T Mutation
[0033] (1) Test samples:
[0034] Twenty-eight children with sensorineural deafness were diagnosed in the 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 test sample, positive control substance or negative control substance to the quantitative PCR reaction solution tube, perform PCR amplification on a two-color (or above) fluorescent quantitative PCR instrument, and FAM and HEX channels are used to detect the T base and C 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
[0038] The first po...
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Abstract
The invention provides a fluorescent quantitative PCR detection kit for mitochondrial deafness C1494T common mutation C1494T. The fluorescent quantitative PCR detection kit comprises a quantitative PCR reaction liquid, a first positive reference substance, a second positive reference substance, a negative reference substance, a direction and a case body, wherein the quantitative PCR reaction liquid contains a PCR buffer solution, MgCl2, dNTPs, heat-proof DNA polymerase, an upstream amplimer, a downstream amplimer, a first fluorescent probe and a second fluorescent probe. Two MGB probes are designed with respective to C>T single base mutation of the 1494 site of mtDNA, the deafness-related mitochondrial 1555 site nucleotide is detected through fluorescent quantitation PCR, and whether the site is subjected to C>T mutation or not is determined. The fluorescent quantitative PCR detection kit has the advantages of being simple, convenient, quick and accurate, can be applied to clinical diagnosis and treatment of mitochondrial C1494T mutation-related deafness and provides references for prevention and treatment of mitochondrial deafness caused by C1494T 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 C1494T 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] Clear literature has shown that the C1494T mutation located in mtDNA 12S rRNA is closely related to amino...
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