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Chip and kit for detecting non-deletion alpha-thalassemia

A thalassemia, non-deletion technology, applied in the field of alpha thalassemia kits, can solve the problems of tedious DNA extraction and easy cross-contamination, and achieve the effects of saving manpower and reagent costs, reducing pollution, and shortening detection time.

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

[0008] The technical problem to be solved by the present invention is: the purpose is to overcome the above-mentioned deficiencies of the prior art, directly carry out PCR amplification with whole blood, amniotic fluid and filter paper dry blood spot, avoid the shortcoming of tedious work and easy cross-contamination of extracting DNA, and The amount of blood samples required is small, and the storage and transportation of samples are also very convenient

Method used

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  • Chip and kit for detecting non-deletion alpha-thalassemia
  • Chip and kit for detecting non-deletion alpha-thalassemia
  • Chip and kit for detecting non-deletion alpha-thalassemia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1: Using the kit specimen source and PCR template type of the present invention

[0070] 1.1 Sample collection and preparation of PCR templates: a) Sample collection: Specimens are derived from anticoagulated peripheral blood, DBS samples, embryonic villus tissue, amniotic fluid, peripheral blood or umbilical cord blood, etc.; b) Preparation of PCR templates: The above samples are directly used as templates , DNA extracted from the above-mentioned specimens can also be used as a template.

[0071] 1.2 DBS sample preparation

[0072] The above 1.1 filter paper dry blood spot sample (DBS) collection and preparation are as follows:

[0073] ①. Use Whatman903 filter paper (or ordinary filter paper). Mark the subject number and date of collection on the filter paper.

[0074] 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, t...

Embodiment 2

[0076] Example 2. Design of specific primers for PCR amplification and determination of PCR reaction system

[0077] 1.1 Primer design

[0078] The α-globin gene sequence was obtained from the GenBank database, and a pair of primers were designed according to the point mutation of the α-thalassemia gene for PCR amplification of the α-thalassemia gene. Primers were synthesized by a professional biological company. Primer sequences are listed in Table 1.

[0079] Table 1 Detection of non-deleted α-thalassemia gene primers and specific oligonucleotide probe sets

[0080]

[0081]

[0082] 1.2 Determination of multiplex PCR reaction system

[0083] Using the orthogonal test method, through a large number of experimental comparisons, the optimal PCR reaction solution formula and system are finally determined as shown in Table 2: PCR reaction solution 18 μ l, peripheral blood (chilli, amniotic fluid or umbilical cord blood) sample volume 2μl, 1-3 pieces of DBS samples (dia...

Embodiment 3

[0089] Example 3: Design, spotting and immobilization of oligonucleotide probes

[0090] 1. Probe design and screening

[0091] The α-globin gene sequence was obtained from the GenBank database, and three pairs of mutants (α CS α, α QS α, α WS α) Corresponding probes, design an oligonucleotide probe combination that specifically recognizes a certain genotype; and design negative (NC), positive (AC), chromogenic control probes (CC) (see Table 1) and blank Control (BC). Synthesized by a professional biological company.

[0092] 3.2. Preparation of gene chip.

[0093] The preparation method of the gene chip of the present invention comprises the following steps:

[0094] (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 solution (10 μM) for sample application.

[0095] (2) Immobilizing the probe: the nylon membrane was activated by soaking in 10% ED...

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Abstract

The invention relates to a gene chip, a PCR amplification reagent and a kit for detecting non-deletion alpha-thalassemia genes, and belongs to a molecular diagnosis technology. According to the present invention, based on direct multiplex PCR, Gap-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 invention relates to a molecular detection of non-deletion type α-thalassemia, in particular to a kit for detecting α-thalassemia caused by point mutation by direct PCR (Direct PCR). Background technique [0002] Thalassemia (thalassemia, referred to as thalassemia), that is, globin anemia, is an autosomal recessive genetic disease. A group of autosomal monogenic hemolytic anemias caused by reduced hemoglobin production. It is more common in Guangdong, Guangxi, and Sichuan in my country. There are sporadic cases of autosomal monogenic hemolytic anemia caused by globin gene deletion or point mutation in the provinces and regions south of the Yangtze River. According to the classification of the amino acid chains involved, thalassemia is usually divided into four types: α, β, δβ, and δ, among which α and β thalassemia are more common. [0003] In 1925, Cooley and Lee first described thalassemia, which was first discovered in the Mediterranean regio...

Claims

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Application Information

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IPC IPC(8): C12Q1/6837
CPCC12Q1/6837
Inventor 陈治中卿吉琳
Owner 陈治中
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