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A barcoded magnetic bead liquid-phase chip detection kit for thalassemia gene

A technology of liquid phase chip detection, thalassemia, applied in the field of molecular biology

Active Publication Date: 2018-05-18
BIOCHAIN BEIJING SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The present invention uses the Intelliplex of barcoded magnetic beads (BMB) TM The technology recognizes the micro-etched barcode on the magnetic beads under visible light, and decodes and classifies the magnetic beads. Since the barcoded magnetic beads themselves do not contain fluorescent substances, it effectively solves the problems existing in the Luminex platform

Method used

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  • A barcoded magnetic bead liquid-phase chip detection kit for thalassemia gene
  • A barcoded magnetic bead liquid-phase chip detection kit for thalassemia gene
  • A barcoded magnetic bead liquid-phase chip detection kit for thalassemia gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1 Preparation of PCR mix and positive control substance for amplifying thalassemia gene target fragment

[0072] 1. Design and synthesis of PCR primers

[0073] According to the distribution of mutation hotspots of thalassemia genes in the population, the genome sequence was downloaded from NCBI, and the amplified product was designed to cover all non-deleted β-globin according to the sequence 1 (gi|568815587:c5227271-5225466Homo sapiens chromosome11, GRCh38.p2PrimaryAssembly) The upstream and downstream specific primer sequences of the gene mutation site; and according to the sequence 2 (gi|568815582:165047-186210Homo sapiens chromosome 16, GRCh38.p2Primary Assembly), the amplification product is designed to cover all non-deletion and deletion α globin gene mutations The upstream and downstream specific primer sequences of the sites were designed, and a Tag sequence with no homology to the human genome was designed. The upstream specific primer sequence of t...

Embodiment 2

[0086] Example 2 Preparation of liquid-phase chip containing loci-specific probes for thalassemia genes

[0087] 1. Probe Design and Synthesis

[0088] Use the NCBI SNP database to determine the distribution of hotspot mutations and specific mutation bases on the thalassemia target gene sequence, design site-specific probes, and design N probes and detection methods for wild templates for each non-deletion site. For positive mutant M probes and deletion sites, one specific probe for detecting amplified products after deletion and one specific probe for detecting wild templates were designed respectively, and the 5' end of the site-specific probes was subjected to Aminolinker C12 Modified, the sequence is complementary to the biotin strand of the PCR amplified product. At the same time, a positive hybridization probe with no homology to the amplified product fragment and a positive hybridization reverse sequence probe complementary to it were designed, the 5' end of the posi...

Embodiment 3

[0102] Example 3 Samples were tested using a thalassemia gene detection kit

[0103] figure 1 Shown is the flowchart of implementing the thalassemia gene detection kit of the present invention, now in conjunction with figure 1 The method for detecting samples by the thalassemia gene detection kit of the present invention will be further described.

[0104] 1. Extraction of human DNA genome

[0105] The extraction of human DNA genome can use commercially available extraction kits or the method in the third edition of the "Molecular Cloning Experiment Guide", and extract the whole blood sample according to the operation manual or the "Molecular Cloning Experiment Guide".

[0106] 2. PCR amplification

[0107] PCR A and PCR B systems were used to amplify the DNA and pure water of the sample to be tested, and the positive control was amplified at the same time. The mutation control A was amplified using the PCR A system, and the deletion control B was amplified using the PCR ...

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Abstract

The invention provides a barcoded magnetic bead liquid phase chip detection kit for thalassemia gene, which includes a PCR reagent for amplifying thalassemia gene and a hybridization reagent for detecting thalassemia gene, wherein the hybridization reagent includes liquid phase chip; the liquid phase chip is composed of barcoded magnetic beads coupled with specific hybridization probes, blank barcoded magnetic beads, free hybridization positive reverse sequence probes and hybridization buffer. The invention has the advantages of simple operation, fast response, high throughput and digitization.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a barcoded magnetic bead liquid phase chip detection kit for thalassemia gene. Background technique [0002] Thalassemia, also known as thalassemia, or thalassemia for short, is the most common human monogenic hereditary blood disease in the world. It is a group of globin peptide chain synthesis reduced or unable to be synthesized due to globin gene deletion or point mutation. hereditary hemolytic anemia. The incidence rate is relatively high in the provinces south of the Yangtze River in my country, among which Guangdong, Guangxi, Sichuan, Yunnan, Hainan, Hong Kong, and Taiwan are more common, and it is rare in the north. [0003] Thalassemia is caused by the diversity of globin deficiency (the type and quantity of the lacking globin chains are different), the clinical manifestations are diverse, and the severity of symptoms is also differ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12Q1/6858
Inventor 贺文妃马竣
Owner BIOCHAIN BEIJING SCI & TECH
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