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Beta-thalassemia mutation detection kit

A technology for thalassemia and mutation detection, which is applied in the field of kits for detecting β-thalassemia nucleotide polymorphisms, and can solve the problems of inability to detect mutations and lengthy hybridization process.

Active Publication Date: 2011-07-06
GUANGZHOU DARUI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Qu Jing et al. disclosed "Nucleic acid hybridization membrane strip and kit for diagnosing β-thalassemia" (publication number: CN1718742A), which only detects 17 mutation types in China; Anemia DNA chip and its preparation method" (publication number: CN 1453363A) can detect 21 types of mutations found in China, but both patents have a common deficiency: they cannot detect 27 types of mutations found in China , and its probe design needs a lot of probe screening to get satisfactory results; both amplify the β-Globin gene into two segments, one of which is about 600bp, and the other is about 400bp
[0012] At present, the commercial reagents based on membrane hybridization at home and abroad all adopt the above principle, and the whole hybridization process is very lengthy.

Method used

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Effect test

Embodiment 1

[0039] The preparation of embodiment 1 sample target nucleic acid

[0040] 1) EDTA anticoagulated whole blood collected from 6 clinical cases, and gently invert the glass tube to mix 5-10 times.

[0041] 2) Take a 1.5ml clean centrifuge tube and mark the tube cap with a Mark pen. Add 300μl of mixed anticoagulant blood into a centrifuge tube, add 1ml of sterilized double distilled water, mix well, and place at room temperature for 3 minutes.

[0042] 3) Centrifuge at 3000 rpm for 5 minutes at room temperature. Discard the supernatant and keep the maroon precipitate.

[0043] 4) Repeat the above steps once. Remove as much residual liquid as possible.

[0044] 5) Add DNA extraction solution (5% Chelex-100) and mix thoroughly. Water bath at 100°C for 10 minutes, centrifuge at 10,000 rpm for 5 minutes, and store at -20°C for later use.

Embodiment 2

[0045] The PCR amplification of embodiment 2 target nucleic acid

[0046] PCR reagents use hot start enzyme or Taq enzyme reaction system, which are hot start enzyme reaction system and Taq enzyme reaction system respectively, wherein Taq enzyme is produced by NEB company, hot start enzyme is produced by Fermentas, 10× hot start PCR buffer and Taq 5×Master Mix is ​​also commercially available, and the preparation of the reaction system is as follows:

[0047]

[0048] For the PCR reaction system, directly add 2 μl of the extracted DNA template, centrifuge briefly (3 seconds), and put each reaction tube into the PCR instrument.

[0049] Hot start enzyme reaction system amplification conditions: 95°C for 15min, then 94°C for 30s, 56°C for 45s, 72°C for 60s, 45 cycles, and finally 72°C for 7min.

[0050] Taq enzyme reaction system amplification conditions: 94°C for 5min, then 94°C for 30s, 56°C for 45s, 72°C for 60s, 45 cycles, and finally 72°C for 7min.

[0051] The amplifi...

Embodiment 3

[0052] Embodiment 3 Preparation of low-density gene chip

[0053] The low-density gene chip carrier is a nylon membrane, and the membrane strip needs to be used with 10% EDC-HCl (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, Sigma company) solution before use Fully soak and activate for 60 minutes, wash with pure water twice for 3 minutes each time, and then dry at room temperature. The freeze-dried powder of the probe was centrifuged at 12000rpm for 5 minutes, then dissolved in sterilized pure water to 10μM, and then 1.0μl per well was directly applied to the appropriate position of the membrane strip in a fixed format, as follows:

[0054] Low Density Gene Chip Probe Array

[0055]

[0056] After spotting the sample, let it stand at room temperature for 2 hours, treat the membrane strip with 0.1M NaOH solution for 10 minutes, wash it thoroughly with distilled water, dry it at room temperature and store it at 4°C for later use.

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Abstract

The invention relates to a beta-thalassemia mutation detection kit, in particular to a kit for detecting the nucleotide polymorphism of beta-thalassemia by an asymmetric amplification method. The kit comprises a set of specific nucleotide polymorphism probes and a polymerase chain reaction (PCR) primer for amplifying a target gene in a sample, and can detect 27 types of beta-thalassemia mutations; and the detection process is quicker, and the detection result is more accurate.

Description

technical field [0001] The present invention relates to a β-thalassemia mutation detection kit, in particular to a kit for detecting β-thalassemia nucleotide polymorphism by using an asymmetric amplification method. The kit contains a set of specific nucleosides Acid polymorphism probes and PCR primers to amplify target genes in samples for detection of beta-thalassemia due to point mutations. Background technique [0002] Thalassemia is divided into α-thalassemia and β-thalassemia (referred to as β-thalassemia). β-thalassemia is a monogenic genetic blood disease caused by the imbalance of peptide chain expression caused by the abnormality of β-globin gene. Most of them are caused by point mutations of β-globin gene. Beta-thalassemia is one of the most common hereditary diseases in the world. According to statistics, more than 100 million people in the world carry the β-thalassemia gene. my country's Guangdong, Guangxi, Sichuan, Hong Kong, northern Taiwan, Yunnan, Guizhou,...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 李明林炳生张帆陈华云程钢
Owner GUANGZHOU DARUI BIOTECH
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