Kit for integrated detection of alpha and beta mutant type thalassemias

A technology of thalassemia and kits, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, etc., and can solve problems such as inability to effectively distinguish, inability to detect mutations, and difficulty in commercialization

Inactive Publication Date: 2011-10-19
DAAN GENE CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The commercialization of these two patents is difficult because they both have common deficiencies: the 27 types of mutations found in China cannot be detected, and the probe design requires a large number of probe screening to obtain satisfactory results; Amplify the β-Globin gene into two segments, one of which is about 600bp, and the other is about 400bp
Guo Xuemin and others disclosed "a method for designing oligonucleotide probes" (publication number: CN 1461811A), which is to design one or more artificial mutations outside the mutation site to form double mutation or multiple mutation sites, But this still cannot effectively distinguish the G.G mismatch formed after C→G

Method used

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  • Kit for integrated detection of alpha and beta mutant type thalassemias
  • Kit for integrated detection of alpha and beta mutant type thalassemias
  • Kit for integrated detection of alpha and beta mutant type thalassemias

Examples

Experimental program
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Effect test

Embodiment 1

[0049] The preparation of embodiment 1 sample target nucleic acid

[0050] 1) EDTA anticoagulated whole blood collected from 6 clinical cases, and gently invert the glass tube to mix 5-10 times.

[0051] 2) Take a 1.5ml clean centrifuge tube and mark the tube cap with a Mark pen. Add 300 μl of anticoagulated whole blood into a centrifuge tube, add 1ml of sterilized double distilled water, mix well, and place at room temperature for 3 minutes.

[0052] 3) Centrifuge at 3000 rpm for 5 minutes at room temperature. Discard the supernatant and keep the maroon precipitate.

[0053] 4) Repeat the above steps once. Remove as much residual liquid as possible.

[0054] 5) Add DNA extraction solution (5% Chelex-100, 0.25ug / mL proteinase K) and mix well. 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

[0055] The PCR amplification of embodiment 2 target nucleic acid

[0056] PCR reagents were prepared using hot start enzyme or Taq enzyme, in which Taq enzyme was produced by NEB Company, hot start enzyme was produced by Fermentas, 10×hot start PCR buffer and Taq 5×Master Mix were also commercially available. Preparation of reaction system as follows:

[0057]

[0058]

[0059]To the PCR reaction reagents, directly add 2 μl of the extracted DNA template, centrifuge briefly (3 seconds), and put each reaction tube into a PCR instrument. The amplification conditions of the PCR reagent with hot start enzyme are: 95°C for 15min, then 94°C for 30s, 68°C for 90s, run 5 cycles, then 94°C for 30s, 55°C for 45s, 72°C for 60s, run for 40 cycles, and finally 72°C for 7min.

[0060] The PCR reagent amplification conditions with Taq enzyme added are: 94°C for 5min, then 94°C for 30s, 68°C for 90s, run 5 cycles, then 94°C for 30s, 55°C for 45s, 72°C for 60s, run for 40 cycles, and fi...

Embodiment 3

[0062] The preparation of embodiment 3 low-density chips

[0063] The low-density chip carrier is a nylon membrane, and the membrane strip needs to be fully diluted with 10% EDC-HCl (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, Sigma Company) solution before use. Soak and activate for 60 minutes, wash with pure water twice, 3 minutes each time, and then dry at room temperature. Centrifuge the freeze-dried powder of the probe at 12000rpm for 5 minutes, then dissolve it in sterilized pure water to 10μM, and then apply 0.5μl per hole directly on the appropriate position of the membrane strip in a fixed format, see attached image 3 .

[0064] 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 kit for integrated detection of alpha and beta mutant type thalassemias, in particular to a kit for detecting alpha and beta thalassemias mutant types by using multiple asymmetric amplification and reverse dot blot hybridization techniques. The kit of the invention consists of a polymerase chain reaction (PCR) reagent, a low-density chip and a hybridizing reagent, and contains a set of specific nucleotide polymorphism probes and PCR primers for amplifying target gene in an amplification sample. The kit can be used for detecting six alpha thalassemia locus mutations and 27 beta thalassemia mutations, which are common in China, at the same time. The detection process of the kit is faster and the detection result of the kit is more accurate, so the kit is expected to be widely used in diagnosis of thalassemia in clinic and instruction on sound child rearing.

Description

technical field [0001] The present invention relates to a kit for the integrated detection of α and β mutant thalassemias, in particular to a kit for detecting α and β thalassemia mutants using multiple asymmetric amplification and reverse dot hybridization techniques. The box contains a set of specific nucleotide polymorphism probes and PCR primers for amplifying the target gene in the sample, which can simultaneously detect α and β thalassemia caused by point mutations, and will be widely used in the detection of thalassemia and prenatal and postnatal care guide. Background technique [0002] Thalassemia is divided into several types such as α, β, δ, δβ, γδβ, etc. The common ones are α-thalassemia (abbreviated as α-thalassemia) and β-thalassemia (abbreviated as β-thalassemia), which are the most common and most harmful in the southern provinces of my country. It is one of the most common genetic diseases in the world. According to WHO statistics, there are more than 150 m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 李明彭春梅林炳生张太松陈华云程钢何蕴韶
Owner DAAN GENE CO LTD
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