Method and kit for simultaneously detecting multiple mutations at hba1/2 and hbb gene loci

A gene site and kit technology, applied in the field of primer combinations for detecting multiple mutations of HBA1/2 and HBB genes, can solve missed and false detections, low accuracy, and inability to simultaneously detect multiple deletion and non-deletion mutations and other problems, to achieve the effect of low false detection rate and wide detection range

Active Publication Date: 2021-06-29
BERRYGENOMICS CO LTD
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  • Summary
  • 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 at hba1/2 and hbb gene loci
  • Method and kit for simultaneously detecting multiple mutations at hba1/2 and hbb gene loci
  • Method and kit for simultaneously detecting multiple mutations at hba1/2 and hbb gene loci

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Example 1: Using the 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 Gene-mutated peripheral blood samples:

[0108]

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

[0110]

[0111] After the amplification was completed, 20ul of each sample was taken and tested on a 1% DNA gel. The results were as follows figure 2 shown, HBA1 / 2 Different deletion mutations in genes and HBB genes can be efficiently amplified.

Embodiment 2

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

[0113] Step 1: Multiplex PCR Amplification

[0114] Prepare a reaction system according to Table 4 below 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 was completed, the amplified product was placed in a centrifuge at 10,000 rpm for 20 min. After centrifugation, it was placed horizontally, and 4 μL of supernatant was added to a new tube.

[0119] Step 2: Construct PacBio Sequencing Libraries

[0120] The reaction system was prepared according to Table 6 below:

[0121]

[0122] On a PCR machine, perform the reaction as follows: 37ºC for 20 min; 25ºC for 15 min; 65ºC for 10 min. After the reaction was completed, 0.5 μL Exonuclease III (NEB,...

Embodiment 3

[0125] Example 3: HBA1 / 2 and HBB Gene mutation detection and validation

[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, People's Hospital of Guizhou Province, Hainan Women and Children's Medical Center, Hunan Home Hui Genetics Specialist Hospital, Jiangxi Maternal and Child Health Hospital, Suining Central Hospital, Xiamen Maternal and Child Health Hospital, and Yunnan Maternal and Child Health Hospital collected peripheral blood of 1759 subjects as verification samples 1759 cases, with reference to Example 2, using the method of the present invention (and kit) simultaneous detection HBA1 / 2 and HBB Multiple mutations at genetic loci. At the same time, the α-thalassemia gene detection kit (Gap-PCR method) of Yaneng Biotechnology (Shenzh...

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Abstract

The present invention relates to a simultaneous detection HBA1 / 2 and HBB A primer set, a kit and a method for multiple mutations of gene loci. The kit includes the following reagents: (1) reagents for multiplex PCR amplification; and (2) reagents for constructing a three-generation sequencing library. Wherein the method comprises the following steps: (1) preparing a subject sample; (2) simultaneously amplifying multiplex PCR in the sample HBA1 / 2 and HBB Gene fragments; (3) Construction of third-generation sequencing library; (4) Sequencing and analysis HBA1 / 2 and HBB Types of genetic mutations.

Description

technical field [0001] The invention relates to a method for simultaneous detection using a three-generation long-reading sequencing platform HBA1 / 2 and HBB Primer combinations and methods for multiple mutations in genes, and kits suitable for this method. Background technique [0002] Hemoglobin (Hb for short) is a special protein that transports oxygen in red blood cells. Adult hemoglobin (HbA) is a tetramer composed of two pairs of α-globin and two pairs of β-globin. Absence or insufficiency of alpha-globin or beta-globin synthesis can lead to dysthalassemia, also known as thalassemia. Loss or insufficiency of α-globin caused by mutations in the α-globin gene is called α-thalassemia (referred to as α-thalassemia); (referred to as β-thalassemia). Thalassaemia is the most common single-gene genetic disease in the world, which is an autosomal recessive inheritance. The high incidence areas include southern China, Southeast Asia, the Mediterranean region, India, the Mid...

Claims

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Application Information

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Patent Type & Authority Patents(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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