Nucleic acid composition for detecting alpha-thalassemia as well as gene chip, kit and application thereof

A technology for thalassemia and nucleic acid composition, applied in the biological field, can solve the problems of cumbersome operation, prone to false negatives, and requiring a large amount of DNA, etc.

Active Publication Date: 2020-07-28
GUANGDONG HYBRIBIO BIOTECH CO LTD +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] Traditional clinical hematological detection methods for α-thalassemia, such as routine parameter detection and analysis, erythrocyte osmotic fragility test and hemoglobin stability test, etc., have low specificity and are prone to false negatives; while peptide chain analysis, HPLC, ELISA and other detection methods Due to single detection and cumbersome operation, it is being replaced by genetic testing with increasingly mature technology
[0013] The traditional genetic diagnosis technology mainly adopts the Southern blot hybridization method, which has high sensitivity and high accuracy, but it is only used for research and cannot be used for large-scale screening because of the long operation time, large amount of DNA required, and the need for isotopes.
Compared with traditional Southern molecular hybridization, ordinary PCR electrophoresis technology is simple, fast, accurate, economical and easy to promote. It provides a more effective method for α-thalassemia gene diagnosis, but its specificity is low and it cannot be detected. Common CS, QS, WS point mutations, and prone to false negative or false positive

Method used

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  • Nucleic acid composition for detecting alpha-thalassemia as well as gene chip, kit and application thereof
  • Nucleic acid composition for detecting alpha-thalassemia as well as gene chip, kit and application thereof
  • Nucleic acid composition for detecting alpha-thalassemia as well as gene chip, kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] This embodiment provides a kit for detecting α-thalassemia, which includes: gene chip, primer pair 1-8, gene chip, PCR buffer, dNTPs, MgCl 2 , Taq enzyme and sterile water for injection.

[0058] The sequences of the primer pairs are shown in Table 1. The gene chip includes a nylon membrane, probes 1-12 immobilized on the nylon membrane, normal control probes corresponding to the deletion α-thalassemia gene, and chromogenic system control probes. The sequences of the probes are shown in Table 2, and the gene chip includes position markers for locating the probes.

[0059] Table 1 Primer pair sequence

[0060]

[0061] Note: The 5' ends of the primers are all labeled with biotin.

[0062] Table 2 Probe sequence

[0063]

[0064]

[0065] Remarks: Probe 13 is the normal control probe corresponding to the deletion α-thalassemia gene, and probe 14 is the control probe for the chromogenic system.

[0066] Preparation of gene chips.

[0067] (1) Spotting and arr...

Embodiment 2

[0077] A detection method for detecting a deletion-type α-thalassemia gene, which comprises using the kit provided in Example 1 to detect a sample to be tested.

[0078] (1) PCR amplification

[0079] The primers in Example 1 were used to perform PCR amplification on the sample to be tested. The PCR reaction solution was 44 μL, the DNA loading volume was 6 μL, and the total reaction volume was 50 μL. The reaction system of PCR amplification is specifically shown in Table 4 and Table 5.

[0080] Table 4 Thalassemia Multiplex PCR Reaction System A

[0081]

[0082]

[0083] Note: Q-solution is an auxiliary reagent for gene amplification.

[0084] Table 5 Thalassemia Multiplex PCR Reaction System B

[0085] Reagent name Added volume (μL) / 1 reaction 10×PCR Buffer 5 5×Q-solution 10 25mM MgCl 2

3 25mM dNTPs 0.8 100 μM upstream primer of primer pair 7 0.2 100 μM downstream primer of primer pair 7 0.2 100 μM upstream pr...

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Abstract

The invention discloses a nucleic acid composition for detecting alpha-thalassemia as well as a gene chip, kit and application thereof, and relates to the technical field of biology. The nucleic acidcomposition comprises a probe combination 1, a probe combination 2 and a probe combination 3, and can be used for simultaneously detecting 10 alpha-thalassemia gene types, namely deletion type alpha-thalassemia gene types (--<SEA>,--[alpha]<3.7>,--[alpha]<4.2>,--<THAI> and --<FIL>), non-deletion type alpha-thalassemia gene types (including three point mutation type alpha-thalassemia gene types ([alpha]<CS>[alpha], [alpha]<QS>[alpha] and [alpha]<WS>[alpha]) and triplet type alpha-thalassemia gene types ([alpha][alpha][alpha]<anti3.7> and [alpha][alpha][alpha]<anti4.2>); the Tm values of probesare similar; hybridization reactions with samples can be performed at the same time; a hybridization result cannot be affected by the temperature problem; and the detection accuracy is high.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a nucleic acid composition for detecting α-thalassemia, its gene chip, its kit and its application. Background technique [0002] Thalassemia (thalassemia for short) is a hemolytic anemia caused by an imbalance in the synthesis rate of α, β-globin peptide chains due to human gene mutation or deletion. Thalassemia is one of the most common genetic diseases in the world, and it is widely distributed in Mediterranean countries and other areas where malaria used to be high. The two common types of thalassemia are α-thalassemia and β-thalassemia, but the carrier rate of α-thalassemia is much higher than that of β-thalassemia. [0003] α-thalassemia is a chronic hemolytic disease caused by autosomal genetic defects, loss or dysfunction of the α-globin gene, resulting in reduced or no synthesis of the α-globin peptide chain. The α-globin gene is located in the α-globin gene cluster at the...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6837C12N15/11
CPCC12Q1/6837C12Q1/6883C12Q2600/156C12Q2565/629
Inventor 谢俊葛毅媛韦薇谢龙旭蓝莹莹杨敏黄嘉慧
Owner GUANGDONG HYBRIBIO BIOTECH CO LTD
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