Gene chip, primer set and kit for single-tube detection of non-deletion alpha-thalassemia and beta-thalassemia

A thalassemia, gene chip technology, used in recombinant DNA technology, microbial assay/inspection, biochemical equipment and methods, etc.

Inactive Publication Date: 2019-01-01
陈治中
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no direct PCR method to detect non-deletion α-thalassemi

Method used

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  • Gene chip, primer set and kit for single-tube detection of non-deletion alpha-thalassemia and beta-thalassemia
  • Gene chip, primer set and kit for single-tube detection of non-deletion alpha-thalassemia and beta-thalassemia
  • Gene chip, primer set and kit for single-tube detection of non-deletion alpha-thalassemia and beta-thalassemia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Embodiment 1: Using the kit specimen source and PCR template type of the present invention

[0103] 1.1 Sample collection and preparation of PCR template: a) Sample collection: Specimens are derived from anticoagulated peripheral blood, DBS samples, villi, amniotic fluid, umbilical cord blood, saliva, genetic material of embryos including gametes such as sperm or eggs, cleavage-stage embryos Blastomeres, blastocyst trophectoderm cells, that is, blastocyst cells, multiple cells or single cells, etc.

[0104] Etc.; b) Preparation of PCR template: the above-mentioned specimen is directly used as a template, and the DNA extracted from the above-mentioned specimen through nucleic acid can also be used as a template.

[0105] 1.2 DBS sample preparation

[0106] The above 1.1 filter paper dry blood spot sample (DBS) collection and preparation are as follows:

[0107] ①. Use Whatman903 filter paper (or ordinary filter paper). Mark the subject number and date of collection on...

Embodiment 2

[0109] Example 2. Design of specific primers for PCR amplification and determination of PCR reaction system

[0110] 1.1 Primer design

[0111] The α-globin and β-globin gene sequences were obtained from the GenBank database, and 3 pairs of primers were designed for PCR amplification according to the mutation regions covered by the α-globin and β-globin genes, and the 3 pairs of primer combinations were placed in the same reaction Multiplex PCR was performed in tubes to simultaneously amplify the α-thalassemia and β-thalassemia genes. Primers were synthesized by a professional biological company. Primer sequences are listed in Table 2.

[0112] Table 2 Amplifies α-thalassemia and β-thalassemia gene primer sets

[0113]

[0114]

[0115] 1.2 Determination of multiplex PCR reaction system

[0116] Using the orthogonal test method, through a large number of experimental comparisons, the optimal PCR reaction solution formulation system is finally determined as shown in T...

Embodiment 3

[0122] Example 3: Design, spotting and immobilization of oligonucleotide probes

[0123] 3.1. Design and screening of probes

[0124] The α-globin and β-globin gene sequences were obtained from the GenBank database, and the α-thalassaemia mutations (QS, CS, WS) and 19 β-thalassemia mutations were designed to specifically identify α-thalassemia mutations and 19 β-thalassemia mutations. Oligonucleotide probe combinations for poor gene mutation; and design negative (NC), positive (AC1, AC2, AC3), chromogenic control probes (CC) and blank control (BC). Synthesized by professional biological company, see Table 4.

[0125] Table 4 detects oligonucleotide probe sets for α-thalassemia and β-thalassemia gene mutations

[0126]

[0127]

[0128] 3.2. Preparation of gene chip.

[0129] The preparation method of the gene chip of the present invention comprises the following steps:

[0130] (1) Preparation of the working solution of the oligonucleotide probe: use the probe diluent ...

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Abstract

The invention relates to a gene chip, a primer set and a kit for single-tube detection of non-deletion alpha-thalassemia and beta-thalassemia, and belongs to a molecular diagnosis technology. According to the present invention, based on the direct multiplex PCR and reverse dot blot combined detection principle, the corresponding amplification primers and the corresponding probes are designed according to the mutation or deletion sites of each genotype, the primer is labeled with biotin, the probe is labeled with amino, a gene chip is used as substrate, the probe is immobilized on the DNA chip,the PCR product amplified by the specific primer is hybridized with the probe, and the diagnosis of thalassemia is performed by interpreting a signal coloring box.

Description

technical field [0001] The present invention relates to the molecular diagnosis technology of α and β thalassemia, in particular to a single tube direct PCR (Direct PCR) combined with RDB method to detect 3 kinds of non-deletion type α thalassemia point mutations and 19 kinds of β thalassemia mutation sites Gene chips, primer sets and kits. Background technique [0002] Thalassemia (thalassemia, referred to as thalassemia), is due to α or β globin gene deletion or mutation leads to globin chain synthesis disorder, resulting in an imbalance in the ratio of α chain to β chain, the relative excess chain is free, which leads to hemoglobin Unstable and redundant globin chains are deposited on the red blood cell membrane, which is prone to oxidative denaturation, changes the permeability and fragility of the red blood cell membrane, and leads to hemolytic anemia. Clinically, it often shows chronic progressive hemolytic anemia with varying symptoms anemia. And according to the ty...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/156C12Q2600/16C12Q2600/166
Inventor 陈治中卿吉琳
Owner 陈治中
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