Thalassemia mutant gene detection primer pair and kit, and application and library construction methods thereof

A technology for thalassemia and gene detection, applied in biochemical equipment and methods, chemical libraries, combinatorial chemistry, etc., can solve the problems of low detection cost, high cost, and few types, and achieve the effect of reducing detection cost and improving throughput

Inactive Publication Date: 2018-11-13
BGI CLINICAL LAB (SHENZHEN) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] All of the above-mentioned thalassemia gene detection methods have shortcomings to varying degrees. For example, the Gap-PCR method can only detect deletion types; among various detection methods for mutation types, PCR-ARMS can detect few types of mutations, and PCR-ASO, Although PCR-RDB and real-time fluorescent quantitative PCR methods are low in detection cost and can quickly detect multiple mutation types, they can

Method used

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  • Thalassemia mutant gene detection primer pair and kit, and application and library construction methods thereof
  • Thalassemia mutant gene detection primer pair and kit, and application and library construction methods thereof
  • Thalassemia mutant gene detection primer pair and kit, and application and library construction methods thereof

Examples

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Effect test

Embodiment 1

[0086] In this example, 23 samples with known results (including no mutations, common mutations, rare mutations and newly discovered mutations) after sanger sequencing were tested, and the test results were all consistent with the known results (the coincidence rate was 100%). The results show that the present invention can accurately detect the mutation of thalassaemia gene, and has the advantages of high throughput, low cost and accuracy.

[0087] The specific implementation is as follows:

[0088] 1. Sample extraction

[0089] The King Fisher automatic extractor was used to extract DNA from 23 blood samples. The main steps are as follows: Take out 3 deep-well plates and 1 shallow-well plate matched with KingFisher automatic extractor, add a certain amount of matching reagents and label them according to the instructions, and place all well plates with reagents in the corresponding ones as required Position, select the program "Bioeasy_200μL BloodDNA_KF.msz" program, press "star"...

Embodiment 2

[0116] In this embodiment, multiple PCR was used to detect 23 samples in Example 1, and the detection results were all consistent with known results (the coincidence rate was 100%). The results show that the present invention can accurately detect the mutation of thalassaemia gene, and has the advantages of high throughput, low cost and accuracy. The specific implementation is as follows:

[0117] 1. Sample extraction

[0118] Same as Example 1.

[0119] 2. PCR amplification

[0120] The 23 DNA samples obtained in the sample extraction step were numbered 1-23 sequentially, and 23 sets of label primers (Tables 1 and 2) were used to respectively amplify 23 DNA samples. The 24th set of label primers was a negative control without added template. The PCR reaction is carried out in a 96-well plate, using multiple PCR, the amplification of the two genes HBA1 and HBB1 is completed in the same tube, and the amplification of the two genes HBA2 and HBB2 is completed in the same tube; each sam...

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Abstract

Disclosed are a thalassemia mutant gene detection primer pair and kit, and application and library construction methods thereof. The thalassemia mutant gene detection primer pair comprises primer sequences shown as SEQ ID NO: 1-8. The thalassemia mutant gene detection primer pair can be applied to simultaneously detecting a number of mutation types and help discover new mutation types, and when combined with second-generation sequencing, can greatly reduce the detecting cost and improve throughput, thereby achieving significant cost advantages and being beneficial to popularized application ofthalassemia mutant gene detection.

Description

Technical field [0001] The present invention relates to the technical field of thalassemia detection, in particular to a primer set, a kit, its application and a library construction method for thalassemia mutant gene detection. Background technique [0002] Thalassemia (hereinafter referred to as thalassaemia) is a common hemolytic single-gene genetic disease that occurs mostly in the Middle East, Central Asia, Africa, Southeast Asia, and South China. The molecular mechanism of thalassaemia is that the defect of the globin gene reduces or lacks the synthesis of one or more of the peptide chains that it encodes, resulting in an imbalance in the proportion of hemoglobin components, which in turn leads to hemoglobin instability. According to different types of defective globin genes, thalassaemia is mainly divided into α-thalassaemia and β-thalassaemia. [0003] Alpha-thalassemia is a hemolytic anemia disease caused by the deletion or mutation of the alpha-globin gene that inhibits ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6806C12N15/10C12N15/11C40B50/06
CPCC12N15/1093C12Q1/6806C12Q1/6883C12Q2600/156C40B50/06C12Q2531/113C12Q2525/191
Inventor 陈仕平张彩芬杨晓琴张现东方俊彬甄贺富张礼茗陈彩粉刘碧湘刘赛军黄晓燕饶斌李映霞褚仲杰张红云刘娜李云
Owner BGI CLINICAL LAB (SHENZHEN) CO LTD
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