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Reagent, method and kit for detecting rare mutation of thalassemia gene

A technology for thalassemia and rare mutations, applied in biochemical equipment and methods, microbiological determination/testing, DNA/RNA fragments, etc., can solve the problems of missed detection of rare mutations, achieve high sensitivity and specificity, mature and simple, Detects effects with high sensitivity

Pending Publication Date: 2022-04-08
FUZHOU ADICON CLINICAL LAB INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current conventional thalassemia detection kits often cause missed detection of rare mutations. Therefore, in clinical testing, the principle of combining hematological phenotype and genotype should be strictly followed. If it is met, the method of globin gene sequencing should be considered to clarify whether there are rare or new thalassemia mutations, so as to avoid missed diagnosis

Method used

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  • Reagent, method and kit for detecting rare mutation of thalassemia gene
  • Reagent, method and kit for detecting rare mutation of thalassemia gene
  • Reagent, method and kit for detecting rare mutation of thalassemia gene

Examples

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Effect test

Embodiment 1

[0054] Operation process of blood / cell / tissue genomic DNA extraction kit (Tiangen Biology):

[0055] (1) Extract tissue DNA from blood: 1) Extract 300 μl of blood and add 900 μl of erythrocyte lysate, mix by inverting, and place at room temperature for 5 minutes, during which time, invert and mix several times. Centrifuge at 12,000 rpm for 1 min, suck off the supernatant, leave the white blood cell pellet, add 200 μl buffer GA, shake until thoroughly mixed; 2) add 20 μl proteinase K solution, mix well; 3) add 200 μl buffer GB, mix thoroughly by inversion 4) Add 200 μl of absolute ethanol, shake and mix well for 15 seconds, at this time flocculent precipitation may appear, briefly centrifuge to remove Remove the water droplets on the inner wall of the tube cover; 5) Add the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (the adsorption column is placed in the collection tube), centrifuge at 12,000rpm for 30 seconds, pour off the ...

Embodiment 2

[0081] Seven peripheral blood samples clinically diagnosed as thalassemia were taken, and the genome was extracted, reagents were prepared and tested according to the method described in Example 2. Add 1 μl of each sample to the detection system PCR reaction solution. At the same time, make positive, negative, and blank controls. Detect with common PCR instrument, the time is 100 minutes.

[0082] Agarose gel electrophoresis identification, electrophoresis as shown in figure 1 As shown, all samples have the target bands, after sequencing comparison, the sequencing comparison results are as follows figure 2 As shown, using Mutation Surveyor V4.0.8 (demo) to analyze the sequencing results, no mutations were detected in the HBA genes of the 7 samples, and IVS-2-666 was detected in the HBB genes of all 7 samples (C>T ) heterozygous mutation, IVS-2-654 (C>T) heterozygous mutation was detected on the HBB gene in 2 samples, and CD17 (AAG>TAG) homozygous mutation was detected on t...

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Abstract

The invention discloses a reagent, a method and a kit for detecting rare gene mutation of thalassemia. The reagent comprises forward and reverse primers for specifically amplifying rare gene mutation of alpha thalassemia and beta thalassemia, an internal reference primer and a sequencing primer, the Sanger sequencing technology is combined, the rare gene mutation of the thalassemia is rapidly and accurately detected, the genotype of a patient can be clearly determined, and the omission ratio of the rare pathogenic mutation and the birth of severe children can be reduced in a high-incidence area of the thalassemia.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to a reagent, method and kit for detecting rare mutations of thalassemia genes. Common PCR combined with Sanger sequencing technology is used to detect rare gene mutations in α and β thalassemia. Background technique [0002] Thalassemia (referred to as thalassemia) is a chronic hemolytic anemia caused by genetic gene deletion or mutation. It can generally be divided into two categories: α and β thalassemia. It is one of the most common genetic diseases in humans and is widely prevalent in the Mediterranean basin. , Middle East, Southeast Asia and Africa. my country's Guangxi, Guangdong, Hainan, Guizhou, Yunnan, Sichuan and other provinces are high incidence areas of thalassemia. α-thalassemia is a thalassemia caused by the mutation of the gene encoding the α-globin chain, which is divided into two types: deletion type and non-deletion type (mainly point mutation...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/686C12Q1/6869C12N15/11
Inventor 陈雪青王淑一
Owner FUZHOU ADICON CLINICAL LAB INC
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