In-vitro diagnostic kit for gene mutation of glucose-6-phosphate dehydrogenase (G6PD) deficiency

A technology of phosphate dehydrogenase and in vitro diagnosis, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., and can solve the problem of patients not being diagnosed in time or misdiagnosed, unable to distinguish heterozygous or homozygous mutations, and missed detection high rate or false positive rate, achieve high clinical application value, reduce economic and mental burden, and detect quickly and effectively

Active Publication Date: 2014-09-03
CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Some of the existing methods such as single-strand conformation polymorphism analysis (SSCP), denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), high performance liquid chromatography (DHPLC), DNA Direct sequencing, etc., although the detection accuracy is high, its clinical application is limited due to factors such as high cost, complicated procedures, and long time-consuming; while some other approved methods, such as the allele-specific amplification m...

Method used

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  • In-vitro diagnostic kit for gene mutation of glucose-6-phosphate dehydrogenase (G6PD) deficiency
  • In-vitro diagnostic kit for gene mutation of glucose-6-phosphate dehydrogenase (G6PD) deficiency
  • In-vitro diagnostic kit for gene mutation of glucose-6-phosphate dehydrogenase (G6PD) deficiency

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] 1. Sample: DNA extracted from human whole blood (50-100ng / u1). Samples 1 and 2 are normal controls, which means the amplification template is derived from normal human blood DNA without the following 6 high-frequency mutations; samples 3 and 4 are homozygous mutants, which means the amplification template is derived from the following 6 The whole blood DNA of a G6PD-deficient patient with a high-frequency mutation, and the mutation is a homozygous mutation; samples 5 and 6 are heterozygous mutations, which means that the amplification template is derived from a G6PD-deficient patient with the following 6 high-frequency mutations Blood DNA, and the mutation is a heterozygous mutation.

[0047] 2. Reagents: Hot Start Green Master Mix, 2×: Contains 2×Green Go Taq Reaction Buffer (pH8.5), 400μM dATP, 400μM GATP, 400μM dCTP, 400μM dTTP, 4mM MgCl2, Nuclease-Free Water, DNA polymerase.

[0048] 3. T-ARMS-PCR reaction system and amplification program

[0049] ①G1388A uses the follo...

Embodiment 2

[0121] 1. Use the reaction system in Table 1 and the amplification program to perform PCR amplification on each sample. The imaging results of agarose gel electrophoresis are as follows Figure 26 Shown. Sample 1-1 is normal DNA, sample 1-2 is A95G mutant DNA, sample 1-3 is G871A mutant G6PD-deficient patient's whole blood DNA, sample 1-4 is A95G and G871A co-mutated G6PD-deficient patient's whole blood DNA.

[0122] Table 1

[0123]

[0124] PCR amplification program

[0125]

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Abstract

The invention provides an in-vitro diagnostic kit for gene mutation of glucose-6-phosphate dehydrogenase (G6PD) deficiency. The kit comprises SEQ IDNO.1-24. Aiming at six high frequency mutation sites of a G6PD gene, the invention provides a quick in-vitro diagnostic kit for gene mutation of G6PD deficiency, wherein the kit is accurate, good in specificity, high in detection rate, low in cost and good in generalization performance and can differentiate heterozygous and homozygous mutations, thereby providing a quick and reliable molecular diagnostic basis for immediately diagnosing and preventing the G6PD deficiency in gene level. The kit can make a definite diagnosis to the patient within the shortest time, treat immediately suiting the case and avoid further deterioration of the pathogenetic condition, so that the kit not only has important social and economic significance in reducing harm of the G6PD deficiency and reducing the economic and spiritual burden of families, but also has high clinical popularization and application value and wide market prospect.

Description

Technical field [0001] The invention relates to a diagnostic kit, which is used for the in vitro diagnosis of glucose-6-phosphate dehydrogenase deficiency gene mutations. Background technique [0002] Glucose-6-phosphate dehydrogenase (Glucose-6-phosphatedehy Drogenase, G6PD) deficiency is a common X-linked incomplete dominant genetic disease and one of the most common enzyme deficiency diseases in humans. The incidence of G6PD deficiency in my country is about 4% to 15%, and it is as high as 40% in some areas. It is common in southwest my country (Chongqing, Sichuan and Guizhou) and several provinces in southern China (Guangdong, Guangxi, Hainan, etc.). [0003] G6PD is the initiation enzyme of pentose phosphate bypass metabolism that exists in all cells and tissues. It provides pentose sugars for nucleic acid synthesis and reduced coenzyme II (NADPH) for various biosynthesis and maintenance of hemoglobin and red blood cell membranes. The stability. G6PD deficiency not only affe...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q1/6883C12Q2600/156C12Q2537/143C12Q2535/137C12Q2545/101
Inventor 黄轶李阳宋利华
Owner CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV
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