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Gene chip, amplification reagent and kit for detecting alpha-thalassemia

A thalassemia and gene chip technology, which is applied in the field of molecular detection of α-thalassemia, can solve the problems of family and social burden, great harm, and inconvenient use of patients.

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

[0010] The above products and methods all have the risk of missed or false detection of α-thalassemia, resulting in the birth of children with moderate to severe thalassemia, which brings a heavy burden to patients, their families and society
At present, there is no kit that can detect 4 deletions and 3 non-deletions directly, quickly and easily at the same time.

Method used

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  • Gene chip, amplification reagent and kit for detecting alpha-thalassemia
  • Gene chip, amplification reagent and kit for detecting alpha-thalassemia
  • Gene chip, amplification reagent and kit for detecting alpha-thalassemia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Embodiment 1: Using the kit specimen source and PCR template type of the present invention

[0092] 1.1 Sample collection and preparation of PCR template:

[0093] a) Sample collection: samples come from anticoagulated peripheral blood, DBS samples, villi, amniotic fluid or umbilical cord blood, etc.;

[0094] b) Preparation of PCR template: the above-mentioned specimen is directly used as a template, or DNA extracted from the above-mentioned specimen by nucleic acid can also be used as a template.

[0095] 1.2 DBS sample preparation

[0096] The above 1.1 filter paper dry blood spot sample (DBS) collection and preparation are as follows:

[0097] ①. Use Whatman903 filter paper (or ordinary filter paper). Mark the subject number and date of collection on the filter paper. ② Both peripheral blood and anticoagulated blood can be used to make dried blood spot samples on filter paper. If a filter paper disc dried blood spot is prepared from peripheral blood, the collec...

Embodiment 2

[0099] Example 2. Design of specific primers for PCR amplification and determination of PCR reaction system

[0100] 1.1 Primer design

[0101] Obtain the α-globin gene sequence from the GenBank database, design multiple pairs of primers according to the mutation region covered by the α-globin gene, and carry out a large number of experiments; preferably, 1 pair of primers is used for PCR amplification of the non-deleted α-thalassaemia gene, preferably Yes, 4 pairs of primers were placed in the same reaction tube for multiplex PCR to simultaneously amplify the deleted α-thalassemia gene. Primers were synthesized by a professional biological company. Primer sequences are listed in Table 1.

[0102] Table 1 The primer set sequence for detecting α-thalassemia gene

[0103]

[0104] 1.2 Determination of multiplex PCR reaction system

[0105] Using the orthogonal test method, through a large number of experimental comparisons, the optimal PCR reaction solution formula is fin...

Embodiment 3

[0114] Example 3: Design, spotting and immobilization of oligonucleotide probes

[0115] 3.1. Design and screening of probes

[0116] The α-globin gene sequence was obtained from the GenBank database, and the α-thalassemia gene mutation region was designed to specifically identify 7 mutations of α-thalassemia (-- SEA 、-- THAI , -α 4.2 , -α 3.7 、α CS α, α QS α, α WSα) oligonucleotide probe combination; and design negative, positive and chromogenic control probes and blank control (BC). Synthesized by a professional biological company (see Table 4).

[0117] Table 4 detects the probe sequence group of α-thalassaemia gene

[0118]

[0119]

[0120] 3.2. Preparation of gene chip.

[0121] The preparation method of the gene chip of the present invention comprises the following steps:

[0122] (1) Preparation of the working solution of the oligonucleotide probe: use the probe diluent (0.5M Na 2 CO 3 and 0.5M NaHCO 3 solution) were mixed and diluted into a working ...

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Abstract

The invention relates to a gene chip, an amplification reagent and a kit for detecting alpha-thalassemia, and belongs to a molecular diagnosis technology, wherein 3 non-deletion (alpha<CS>alpha, alpha<QS>alpha and alpha<WS>alpha) alpha-thalassemia and 4 deletion (--<SEA>, --<THAI>, -alpha<4.2> and -alpha<3.7>) alpha-thalassemia can be rapidly and simultaneously detected by combining direct PCR andreverse dot blot (RDB).

Description

technical field [0001] The present invention relates to the molecular detection technology of α-thalassemia, in particular to a kind of rapid and simultaneous detection of α-thalassemia three non-deletion types (α CS α, α QS α, α WS α) and 4 deletion types (-- SEA 、-- THAI , -α 4.2 and-alpha 3.7 )Reagent test kit. Background technique [0002] Thalassemia (thalassemia, referred to as thalassemia) is due to the defect of the globin gene (gene mutation or deletion), which reduces or fails to synthesize one or more globin peptide chains of hemoglobin (Hb), which causes hemoglobin. The unbalanced ratio of α-chain / non-α-chain leads to the instability of hemoglobin, the destruction of red blood cells, and the reduction of hemoglobin production. It is a group of the most common lethal and disabled hereditary blood diseases in the world that seriously threaten human health. The main types are alpha and beta thalassemia. [0003] α-thalassemia (hereinafter referred to as α-th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883
CPCC12Q1/6883C12Q2600/156C12Q2600/16C12Q2600/166
Inventor 陈治中卿吉琳
Owner 陈治中
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