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Method and kit for simultaneously detecting multiple mutations of HBA1/2 and HBB gene loci

A gene locus and kit technology is used in the field of primer combinations for detecting multiple mutations of HBA1/2 and HBB genes, which can solve the problems of missed detection and false detection, low accuracy, and inability to determine whether the mutation is linked or not, and achieves detection. The effect of wide range and low detection false detection rate

Active Publication Date: 2021-04-27
BERRYGENOMICS CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The purpose of the invention is to solve the present stage HBA1 / 2 and HBB Gene mutation detection has low accuracy and cannot detect multiple deletion and non-deletion mutations at the same time, it is impossible to determine whether mutations are linked, it is impossible to detect uncommon mutations, and there are problems such as missed detection and false detection in clinical practice

Method used

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  • Method and kit for simultaneously detecting multiple mutations of HBA1/2 and HBB gene loci
  • Method and kit for simultaneously detecting multiple mutations of HBA1/2 and HBB gene loci
  • Method and kit for simultaneously detecting multiple mutations of HBA1/2 and HBB gene loci

Examples

Experimental program
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Effect test

Embodiment 1

[0106] Embodiment 1: Utilize multiplex PCR method of the present invention to amplify different HBA1 / 2 and HBB Gene mutation

[0107] Prepare the reaction system according to the following table 2, and amplify different types HBA1 / 2 and HBB Peripheral blood samples with genetic mutations:

[0108]

[0109] On the PCR instrument, perform pre-amplification according to the conditions shown in Table 3 below:

[0110]

[0111] After the amplification is completed, take 20ul of each sample and detect it on a 1% DNA gel. The results are as follows: figure 2 as shown, HBA1 / 2 Different deletion mutations of genes and HBB Genes can be amplified efficiently.

Embodiment 2

[0112] Example 2: Construction of a PacBio sequencing library using the multiplex PCR method involved in the present invention

[0113] Step 1: Multiplex PCR Amplification

[0114] Prepare the reaction system according to the following table 4 to amplify peripheral blood samples with different types of HBA1 / 2 and HBB gene mutations:

[0115]

[0116] On the PCR machine, perform pre-amplification according to the conditions shown in Table 5 below:

[0117]

[0118] After the amplification is completed, put the amplification product into a centrifuge, centrifuge at 10000rpm for 20min. After the centrifugation, place it horizontally, take 4 μL of the supernatant and add it to a new tube.

[0119] Step 2: Construct the PacBio sequencing library

[0120] Prepare the reaction system according to the following table 6:

[0121]

[0122] On the PCR machine, react according to the following conditions: 37ºC for 20 min; 25ºC for 15 min; 65ºC for 10 min. After the reaction ...

Embodiment 3

[0125] Example 3: HBA1 / 2 and HBB Detection and verification of genetic mutations

[0126] From Changsha Maternal and Child Health Hospital, First Affiliated Hospital of Chongqing Medical University, First Affiliated Hospital of Guangxi Medical University, People's Hospital of Guangxi Tibet Autonomous Region, Third Affiliated Hospital of Guangzhou Medical University, Guizhou Provincial People's Hospital, Hainan Women and Children's Medical Center, Hunan Family Hui Genetic Specialist Hospital, Jiangxi Maternal and Child Health Care Hospital, Suining Central Hospital, Xiamen Maternal and Child Health Care Hospital, and Yunnan Maternal and Child Health Care Hospital collected peripheral blood from 1759 subjects as verification samples for 1759 cases, referring to Example 2, using the method of the present invention (and kit) simultaneous detection HBA1 / 2 and HBB Multiple mutations at gene loci. At the same time, it was detected by the α-thalassemia gene detection kit (Gap-PC...

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Abstract

The invention relates to a primer group, kit and method for simultaneously detecting multiple mutations of HBA1 / 2 and HBB gene loci. The kit comprises the following reagents of (1) a reagent for multiplex PCR amplification; and (2) a reagent for constructing a third-generation sequencing library. The method comprises the following steps of (1) preparing a subject sample; (2) simultaneously amplifying HBA1 / 2 and HBB gene segments in the sample by multiple PCR; (3) constructing the third-generation sequencing library; and (4) sequencing and analyzing HBA1 / 2 and HBB gene mutation types.

Description

technical field [0001] The invention relates to a method for simultaneous detection using a three-generation long-read sequencing platform HBA1 / 2 and HBB A primer combination and method for multiple mutations of a gene, and a kit suitable for the method. Background technique [0002] Hemoglobin (Hb for short) is a special protein that transports oxygen in red blood cells. Adult Hemoglobin (HbA for short) is a tetramer composed of two pairs of α-globin and two pairs of β-globin. Absent or insufficient synthesis of alpha-globin or beta-globin results in thalassemia, also known as thalassemia. Alpha-globin deficiency or deficiency caused by mutations in the alpha-globin gene is called alpha-thalassemia (abbreviated as alpha-thalassemia); beta-globin deficiency or deficiency caused by mutations in the beta-globin gene is called beta-thalassemia (referred to as β-thalassemia). Thalassemia is the most common single-gene genetic disease in the world. It is an autosomal recess...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6869C12N15/11
CPCC12Q1/6883C12Q1/6869C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2535/122
Inventor 毛爱平张晓杰张文琦张建光
Owner BERRYGENOMICS CO LTD
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