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Fluorescence quantitative PCR detection kit for beta-thalassemia mutation

A thalassemia detection kit technology, applied in the field of molecular biology, can solve problems such as false positives, difficulty in popularization, poor PCR amplification, etc.

Inactive Publication Date: 2013-01-02
广州达健生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, PCR combined with specific oligonucleotide probe (PCR-ASO) dot hybridization method can only detect one mutation at a time. Generally, the diagnosis of unknown samples usually requires multiple repeated detections, so it is quite time-consuming and laborious.
The reverse hybridization (RDB) method has poor PCR amplification, too weak probe on the hybridized membrane strip or insufficient washing of the membrane strip, which will lead to false positive or false negative results.
Mutant oligonucleotide extension amplification PCR technology is more suitable for detecting β-thalassemia with known DNA point mutations, and electrophoresis is required, which is time-consuming and laborious
The gene chip method is simple, fast, microquantified, automated, and a new method that can screen more or even all known mutation types in parallel at one time, but it needs to purchase expensive equipment for detection, and it is difficult to popularize

Method used

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  • Fluorescence quantitative PCR detection kit for beta-thalassemia mutation
  • Fluorescence quantitative PCR detection kit for beta-thalassemia mutation
  • Fluorescence quantitative PCR detection kit for beta-thalassemia mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1: Detection of base deletion mutations (CD41-42 deletion mutations) corresponding to amino acids 41 / 42 of the β-globin gene

[0084] Sample description: Positive samples were collected from the wax block tissue of the specific site mutation of β-thalassemia diagnosed clinically and pathologically in the Department of Pathology, Cancer Hospital Affiliated to Sun Yat-sen University. Genomic DNA was extracted from the wax blocks for the following experimental applications.

[0085] Design a pair of probes and common primers that can specifically detect the base deletion corresponding to amino acid 41 / 42 of the β-globin gene:

[0086] Wherein, the sequence of the PCR forward primer is shown in SEQ ID NO:1, and the sequence of the PCR reverse primer is shown in SEQ ID NO:2:

[0087] A1: 5'-CATAACAGCATCAGGAGTGGACAG-3' (SEQ ID NO: 1);

[0088] A2: 5'-ACTGACTCTCTCTGCCTATTGGTCTATT-3' (SEQ ID NO: 2);

[0089] Fluorescent probes are:

[0090] A3: 5'-HEX-AGGACTCAAAGAAC...

Embodiment 2

[0097] Example 2: Detection of the 654th base mutation (IVS-II-654(C→T)) of the second intron of the β-globin gene

[0098] Design a probe and a pair of common primers that can specifically detect the 654th base mutation of the second intron of the β-globin gene:

[0099]Wherein, the sequence of the PCR forward primer is shown in SEQ ID NO:5, and the sequence of the PCR reverse primer is shown in SEQ ID NO:6:

[0100] B1: 5'-TGGTAGCTGGATTGTAGCTGCTATTA-3' (SEQ ID NO: 5);

[0101] B2: 5'-TTGCACCATTCTAAAGAATAACAGTGA-3' (SEQ ID NO: 6);

[0102] Fluorescent probes are:

[0103] B3: 5'-HEX-TGCTATTGCCTTAACC-MGB-3' (SEQ ID NO: 7);

[0104] B4: 5'-FAM-TTGCTATTACCTTAACCC-MGB-3' (SEQ ID NO: 8);

[0105] Among them, the probe B3 that detects the base corresponding to the 654th position of the second intron of the wild-type β-globin gene: 5'-HEX-TGCTATTGCCTTAACC-MGB-3' (SEQ ID NO: 7), detects β- Probe B4 for the mutation of base 654 in the second intron of the globin gene: 5'-FAM-TTGC...

Embodiment 3

[0110] Example 3: Detection of the base mutation corresponding to the 17th amino acid of the β-globin gene (CD17(A→T))

[0111] Design a pair of probes and common primers that can specifically detect the base mutation corresponding to the 17th amino acid of the β-globin gene:

[0112] The sequence of the PCR forward primer is shown in SEQ ID NO: 9, and the sequence of the PCR reverse primer is shown in SEQ ID NO: 10:

[0113] C1: 5'-CTCCTTAAACCTGTCTTGTAACCTTGATA-3' (SEQ ID NO: 9);

[0114] C2: 5'-TGAGGAGAAGTCTGCCGTTACTG-3' (SEQ ID NO: 10);

[0115] Fluorescent probes are:

[0116] C3: 5'-HEX-CCACGTTCACCTTGCCCCACAG-TAMRA-3' (SEQ ID NO: 11);

[0117] C4: 5'-FAM-CCACGTTCACCTAGCCCCACAGG-TAMRA-3' (SEQ ID NO: 12);

[0118] Among them, the probe C3 that detects the base corresponding to the 17th amino acid of the wild-type β-globin gene: 5'-HEX-CCACGTTCACCTTGCCCCACAG-TAMRA-3' (SEQ ID NO: 11), detects the 17th position of the β-globin gene Probe C4 for the base mutation correspon...

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Abstract

The invention relates to a fluorescence quantitative PCR detection kit for beta-thalassemia mutation. The kit comprises a PCR mixing reaction liquid, a positive control and fluorescence probes for detecting beta-thalassemia mutation genotype, wherein the PCR mixing reaction liquid contains PCR primers for amplifying a gene fragment on which a mutation site is positioned, and the mutation site is at least one mutation site selected from deletion mutation of a base corresponding to the site 41 / 42 amino acid of beta-globin gene, C-to-T mutation of a base corresponding to the site 654 amino acid of the second intron of beta-globin gene, A-to-T mutation of a base corresponding to the site 17 amino acid of beta-globin gene, A-to-G mutation of a base corresponding to the site 28 amino acid on the upstream of the promoter of beta-globin gene, A base insertion mutation of a base corresponding to the site 71 / 72 amino acid of beta-globin gene, and G-to-C mutation of a base corresponding to the site 5 amino acid of the first intron of beta-globin gene. With the technical scheme of the present invention, rapid, accurate and high sensitivity detection of mutation conditions of beta-thalassemia gene can be achieved, and especially 6 special site mutations of beta-thalassemia gene can be detected, wherein the 6 special site mutations are common in Chinese.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a fluorescent quantitative PCR kit for detecting gene mutations, in particular to a fluorescent quantitative PCR detection kit for beta-thalassemia mutations. Background technique [0002] Thalassemia (thalassem ia) referred to as "thalassemia", is one of the most common blood genetic diseases in the world. The clinical phenotypes of thalassemia are diverse, ranging from mild to severe, from normal to repeated blood transfusions, and even life-threatening, leading to premature death or stillbirth in minors, which brings huge mental and economic burdens to families and society. Moreover, there is currently no effective medical treatment; therefore, controlling and reducing the birth rate of children with thalassemia can effectively monitor the occurrence of such diseases. Through population screening and genetic counseling, prenatal diagnosis and genetic diagnosis of fetuses born...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 邵琦
Owner 广州达健生物科技有限公司
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