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Method and kit for detecting southeast Asia deletion alpha-thalassemia

A technology of thalassemia and detection method, which is applied in the field of detection of deletional α-thalassemia in Southeast Asia, which can solve the problems of long experimental period, unfavorable clinical promotion and application, and cumbersome operation

Active Publication Date: 2011-08-10
SHENZHEN KANGMEI BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The classic Southern hybridization technique can diagnose Hb Bart's edema fetus, but it is not conducive to clinical application due to many limitations such as cumbersome operation, long experimental period, and the use of radionuclide labels.

Method used

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  • Method and kit for detecting southeast Asia deletion alpha-thalassemia
  • Method and kit for detecting southeast Asia deletion alpha-thalassemia
  • Method and kit for detecting southeast Asia deletion alpha-thalassemia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Synthesis and purification of primers and probes

[0056] Using the α-globin gene with Genebank accession number AE006462 as the target sequence for PCR amplification, the following primers were designed and synthesized:

[0057] Fd5: the nucleotide sequence shown in SEQ ID NO: 1;

[0058] Rd3: the nucleotide sequence shown in SEQ ID NO: 2;

[0059] Rq5: the nucleotide sequence shown in SEQ ID NO: 3;

[0060] The following Taqman probes were synthesized according to the sequence design between the primers:

[0061] Prob-D: 5'-VIC-TTGGCCAGGTGCCGTGGCTT-HBQ-3';

[0062] Prob-Q: 5'-FAM-CCGGGATTCGGACGTCAGGC-HBQ-3'.

[0063] The synthesized primers were purified by PAGE, and the synthesized probe sequences were purified by HPLC. The fluorescent probe Prob-Q uses FAM as the reporter fluorescence; HBQ is used as the quencher; the fluorescent probe Prob-D uses VIC as the reporter fluorescence; HBQ is used as the quencher.

Embodiment 2

[0064] Example 2: Primer Specificity and Sensitivity Analysis

[0065] Using the Southeast Asia deletion homozygous genome as a template to carry out PCR amplification with the primer set having the primer Fd5 of the nucleotide sequence shown in SEQ ID NO: 1 and the primer Rd3 having the nucleotide sequence shown in SEQ ID NO: 2 , for gel electrophoresis detection see image 3 , using the normal human genome as a template to carry out PCR amplification with the primer set of the primer Fd5 having the nucleotide sequence shown in SEQ ID NO: 1 and the primer Rq5 having the nucleotide sequence shown in SEQ ID NO: 3, gel Electrophoresis detection see Figure 4 . Depend on image 3 and Figure 4 As shown, the gel electrophoresis detection of primers Fd5 and Rd3 and the amplification products of primers Fd5 and Rq5 were all single bands, and fewer primer dimers were generated during amplification, indicating good primer specificity.

[0066] The amplified products of primers Fd...

Embodiment 3

[0068] Embodiment 3: Construction of reference plasmid

[0069] 1. Preparation of normal human gene amplification product plasmid control substance:

[0070] Collect normal samples, and use commercial whole blood genome purification reagents to extract the human genome in normal samples. A normal human genome sample is amplified with a primer pair of Fd5 and Rq5, and the obtained PCR product is cloned into a T-vector plasmid. The plasmid was transformed into Escherichia coli JM109, screened and cultivated in LB medium containing ampicillin, and a single clone was selected and cultured by shaking. Extract the plasmid, after purification, detect the concentration, calculate the copy number, and use it as a normal human gene amplification product plasmid reference substance for future use.

[0071] 2. Preparation of plasmid reference substance for homozygous gene amplification product of Southeast Asia deletion:

[0072] Samples of deletional α-thalassemia in Southeast Asia we...

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Abstract

The invention relates to the field of biotechnology, and discloses a method and kit for detecting southeast Asia deletion alpha-thalassemia. The detection method comprises the following steps of: designing a pair of primers Fd5 and Rd3 and a probe Prob-D at two sides of the broken end of a southeast Asia deletion alpha-thalassemia deleted fragment, designing a reverse primer Rq5 and a probe Prob-Q at the 5' end of the deleted fragment, extracting the DNA of a sample to be detected, performing real-time fluorescence PCR (polymerase chain reaction) by taking the DNA of the sample to be detectedas a template, and then analyzing the sample to be detected according to the real-time fluorescence PCR amplification curve. The kit disclosed by the invention comprises the primers Fd5, Rd3 and Rq5 and the probes Prob-D and Prob-Q marked with different wavelength reporting fluorophores. According to the detection method disclosed by the invention, amplification and detection are simultaneously performed according to the fluorescence signal intensity, thus saving time, improving the sensitivity, and being a simple and quick detection method with high sensitivity and easiness in popularization.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method and kit for detecting Southeast Asian deletion α-thalassemia. Background technique [0002] Thalassemia, also known as thalassemia, got its name because it was first discovered in countries along the Mediterranean Sea, such as Italy, Greece, and Malta. It is a group of inherited hemolytic anemias caused by deletions or point mutations in the globin gene. There are four types of peptide chains that make up globin, namely α, β, γ, and δ chains, which are encoded by their corresponding genes. The deletion or point mutation of these genes can cause obstacles to the synthesis of various peptide chains, resulting in the formation of hemoglobin components. Change. Thalassemia is usually divided into four types: α, β, δβ, and δ, among which α-thalassemia is one of the most important types of thalassemia and one of the most common genetic diseases in the world. Southern my country...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 丁亚平毕少辉
Owner SHENZHEN KANGMEI BIOTECH
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