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Kit for diagnosing gene of Leber optic neuropathy in heredity and detecting method

A technology for optic neuropathy and genetic diagnosis, applied in the field of genetic diagnosis of genetic diseases

Inactive Publication Date: 2005-08-31
FUJIAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Retrieval of relevant materials including Chinese patents shows that using whole blood sample allele-specific multiplex PCR technology to simultaneously detect 3 primary mutation sites in LHON mitochondrial DNA by single-tube one-time PCR has not yet been reported.

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  • Kit for diagnosing gene of Leber optic neuropathy in heredity and detecting method

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Effect test

Embodiment 1

[0045] Embodiment 1: the genetic diagnosis kit of Leber's hereditary optic neuropathy of the present invention

[0046] The kit consists of (400 reactions):

[0047] 1.TaKaRa Ex Taq (5U / μl) 50μl

[0048] 2.10×Ex Taq Buffer (Mg 2+ Plus) 1ml

[0049] 3. dNTP Mixture (each 2.5mM) 800μl

[0050] 4.MAS PCR primers mixture (each 20pM) 600μl

[0051] 5.6×Loading Buffer 1ml

[0052] Composition and DNA sequence of MAS PCR primers in the above table

[0053] Primer name Nucleic acid position Primer sequence

[0054] MS11778F 11759~11778nt 5'-TACGAACGCACTCACAGACA-3'

[0055] MS14484F 14460~14484nt 5′-CTGTAGTATATCCAAAGACAACGAC-3′

[0056] MS3460F 3441~3460nt 5'-ACTACAACCCTTCGCTGTCA-3'

[0057] 11778R 11961~11980nt 5'-GGAGTATAGGGCTGTGACTA-3'

[0058] 14484R 14734~14753nt 5'-GGGTCATTGGTGTTCTTGTA-3'

[0059] 3460R 3781~3800nt 5'-AGGGTGGAGAGGTTAAAGGA-3'

Embodiment 2

[0060] Example 2: Detection method for genetic diagnosis of Leber's hereditary optic neuropathy

[0061] Use the test kit of embodiment 1, carry out according to the following steps:

[0062] 1) Prepare the MAS PCR reaction system (25μl) according to the table below, mix well and place on the PCR machine;

[0063] 10×Ex Taq Buffer (Mg 2+ Plus) 2.5μl

[0064] dNTP Mixture (2.5mM each) 1.0μl

[0065] MAS PCR primers mixture (each 20pM) 1.5μl

[0066] Whole blood sample 0.5μl

[0067] TaKaRa Ex Taq (5U / μl) 0.13μl

[0068] dH 2 O 19.37 μl

[0069] 2) Carry out PCR reaction according to the following conditions:

[0070] Pre-denaturation at 94°C for 4min→30 cycles at 94°C for 30sec→4min at 72°C→storage at 4°C

[0071] 59.8℃ 30sec

[0072] 72℃ 45sec

[0073] 3) After the reaction, add 5 μl 6×Loading Buffer to mix, and take 10-15 μl of 1.5%-1.8% agarose gel electrophoresis for 15-20 minutes and then observe on the ultraviolet instrument. ...

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Abstract

A reagent kit for the gene diagnosis of Leber's hereditary optical nerve (LHON) lesion which is caused by the mutations on 3 primary pathogenic sites (G11778A, G3460A and T14484C) is disclosed. Its detecting process includes such steps as designing and synthesizing the site specific PCR primer, using whole blood as DNA template, PCR amplification, and detecting said mutant sites of LHON patient.

Description

technical field [0001] The invention relates to a method for using genes to diagnose hereditary diseases, in particular to a genetic diagnosis kit for Leber's hereditary optic neuropathy and a detection method thereof. Background technique [0002] In 1871, German ophthalmologist Theodor Leber first reported a family of hereditary optic neuropathy, mainly manifested as acute or subacute bilateral central vision loss, only passed through the maternal line, often involving young males, now known as Leber's hereditary optic neuropathy (Leber's hereditary optic neuropathy) hereditary optic neuropathy, LHON). In 1988, Wallace et al. first discovered that LHON was associated with mitochondrial DNA (mtDNA) G11778A mutation. So far, there are more than 40 related mtDNA mutations reported, of which 90%-95% of LHON patients are associated with three primary mtDNA mutations (G11778A, G3460A and T14484C) related. The distribution of various primary pathogenic mutations is different in...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 阳菊华童绎陈贻锴林宇岚
Owner FUJIAN MEDICAL UNIV