Deletion beta thalassemia detection primers and beta thalassemia detection kit based on SNP analysis and application of deletion beta thalassemia detection kit

A thalassemia and detection kit technology, applied in the field of genetic engineering, can solve the problems of high requirements on instruments and equipment, unsuitable for routine application in grassroots units, cumbersome operation, etc., to achieve the effect of optimizing the reaction system and conditions

Active Publication Date: 2019-12-27
GUANGDONG WOMEN & CHILDREN HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods are relatively cumbersome to operate and require high equipment conditions, which are not suitable for routine application in grassr...

Method used

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  • Deletion beta thalassemia detection primers and beta thalassemia detection kit based on SNP analysis and application of deletion beta thalassemia detection kit
  • Deletion beta thalassemia detection primers and beta thalassemia detection kit based on SNP analysis and application of deletion beta thalassemia detection kit
  • Deletion beta thalassemia detection primers and beta thalassemia detection kit based on SNP analysis and application of deletion beta thalassemia detection kit

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Embodiment 1

[0054] The present invention establishes a method for detecting deletion mutation of β-globin gene cluster based on SNP analysis. The present invention screens out a group of six SNP sites for detection of beta globin gene cluster deletion mutations through bioinformatics analysis and sequencing data of a large number of population samples. Based on the principle of four-primer amplification hindered mutation PCR, specific primers for the above SNP sites were designed, and the typing detection of the six SNP sites was integrated into two multiplex PCR systems. At the same time, the primers were optimized, and the multiplex PCR amplification reaction system was optimized.

[0055] The genome template used in the present invention is obtained by extracting the genome from peripheral blood, and the peripheral blood samples are collected from Guangdong Province.

[0056] (1) SNP site screening

[0057] Based on the principle that the deletion of DNA fragments can lead to the los...

Embodiment 2

[0106] Peripheral blood samples from 10 patients with deletion mutations in the β-globin gene cluster were taken, including Chinese G γ( Aγδβ) 0 3 cases of -Thal deletion type, 2 cases of Yunnanese deletion type, 2 cases of Cantonese deletion type, 3 cases of (SEA)-HPFH deletion type; another 5 normal human peripheral blood samples without β-globin gene cluster deletion mutation were collected, The gene mutation types of all samples were verified by Gap-PCR or MLPA. The DNA of the above 15 samples was extracted and amplified with the following system, and the SNP typing results were compared with the sequencing method.

[0107] 3.1. Preparation of Multiplex PCR Amplification System

[0108] Reaction system A: The composition of each 50 μL reaction system is as follows: Premix LA Taq TM Master mix 25 μL, primers SNP1-in-F 0.2 μL, SNP1-in-R 1 μL, SNP3-in-F 0.2 μL, SNP3-in-R 1 μL, SNP5-in-F 1 μL, SNP5-in-R at a concentration of 10 μM 0.6 μL, 1 μL of OUT-F and 1 μL of OUT-R; ...

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Abstract

The invention discloses deletion beta thalassemia detection primers and a beta thalassemia detection kit based on SNP analysis and application of the deletion beta thalassemia detection kit. The sequences of the detection primers are shown as SEQ ID NO.1-14. The detection kit provided by the invention comprises the primers. The detection primers refer to six SNP loci, after a to-be-detected sampleis subjected to PCR amplification, whether beta-globin gene cluster deletion mutation occurs or not is judged according to whether heterozygous types of the sample SNP genotypes exist or not. If theexperiment result shows that heterozygous loci exist in 6 SNPs of the sample, nine kinds of beta-globin gene cluster deletion mutation can be excluded. Otherwise, if the typing detection results of the six SNP loci are all homozygotic types, the sample is quite likely to be subjected to beta-globin gene cluster deletion mutation, resulting in loss of heterozygosity, and it is prompted that furtherclinical detection needs to be conducted. Typing detection to the SNPs by the detection primer and kit is easy and convenient to operate, and the cost is low.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a detection primer, kit and application of deletion-type β-thalassemia based on SNP analysis. Background technique [0002] Thalassemia is one of the most common monogenic genetic diseases. Common types of thalassemia include α-thalassemia and β-thalassemia, which are caused by abnormal expression of α-globin gene cluster and β-globin gene cluster, respectively. It is mainly distributed in Southeast Asia. It is estimated that about 1.5% of the world's population (800,000 to 90,000,000 people) are carriers of thalassemia, and at least 60,000 newborns are seriously affected every year, accounting for the vast majority in developing countries. In my country, the area south of the Yangtze River is a high-incidence area of ​​thalassemia, mainly in Guangxi, Guangdong, and Hainan. At present, there is no effective treatment for this disease. Children with severe...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6883C12Q1/6858C12Q2600/156C12Q2600/16C12Q2531/113C12Q2535/137C12Q2537/143
Inventor 骆明勇胡思琪王继成杨笑涵詹文丽谢佳陈柯艺胡听听杜丽尹爱华
Owner GUANGDONG WOMEN & CHILDREN HOSPITAL
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