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Target sequence complementary quenching probe and kit for detecting beta globin gene point mutation

A β-globin and detection reagent technology, which is applied in the field of target sequence complementary quenching probes for detecting β-globin gene point mutations and a kit thereof, and can solve problems such as inability to distinguish accurately

Active Publication Date: 2017-11-24
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, after 2-3 years of clinical application, the limitations of the kit’s technical scheme and detection system have also been highlighted. Some mutation sites that are more common in southern China are not within the detection range, such as Initiation CD (ATG>AGG), CAP+ 1(A>C), etc., and adjacent mutation sites such as -28(A>C) and -29(A>G), CD41 / 42(-CTTT) and CD43(G>T) are not accurate distinguish

Method used

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  • Target sequence complementary quenching probe and kit for detecting beta globin gene point mutation
  • Target sequence complementary quenching probe and kit for detecting beta globin gene point mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 A kit for detecting point mutations in the β globin gene

[0073] For the detection of β globin genes -32(C>A), -31(A>C), -30(T>C), -29(A>G), -28(A>G), Cap+1 (A>C), Int(ATG>AGG), CD14 / 15(+G), CD17(A>T), CD26(G>A), CD27 / 28(+C), CD30(A>G), IVS-1-1(G>T), IVS-1-5(G>C), CD31(–C), CD37(G>A), CD41 / 42(-TCTT), CD43(G>T), Target Sequence Complementary Quenching Probe Kit for CD71 / 72(+A), CD71 / 72(+T), IVS-2-654(C>T) Point Mutations

[0074] Kit composition:

[0075] (1) 2 pairs of primers for specific enrichment and amplification of β globin gene mutation hotspot sequence

[0076] 1F: 5'-gccaaggacaggtacggctgtcatc-3' (SEQ ID NO: 1);

[0077] 1R: 5'-ggcaaaggtgcccttgaggttgtc-3' (SEQ ID NO: 2);

[0078] 2F: 5'-cagggcaataatgatacaatgtatcatgc-3' (SEQ ID NO: 3);

[0079] 2R: 5'-gctgtgggaggaagataagaggtatgaac-3' (SEQ ID NO: 4);

[0080] (2) Target sequence complementary quenching probe

[0081] Complementary Quencher Probes for Specific Detection of Wild-type Targets at Po...

Embodiment 2

[0151] Example 2 A kit for detecting point mutations in the β globin gene

[0152] The kit of the present invention is divided into two reaction tubes according to the fluorescent label and melting temperature of the detection probe. The sites and genotypes detected by the probes in reaction tube 1 and reaction tube 2 are shown in Table 1 below.

[0153] Table 1 Sites and genotypes detected by probes in reaction tube 1 and reaction tube 2

[0154]

[0155]

Embodiment 3

[0156] Example 3 A method for detecting point mutations in the β globin gene

[0157] The using method of detection system of the present invention is:

[0158] (1) Sample processing:

[0159] Dilute gDNA samples to 10-100ng / μl with sterilized double distilled water for later use. Among them, gDNA samples can be obtained by the following methods: extraction of peripheral whole blood samples, anticoagulation with EDTA, extraction of gDNA samples by using Tiangen column peripheral blood genomic DNA column extraction reagent (Beijing Tiangen Biotechnology Company); or using traditional phenol The gDNA samples were obtained by chloroform extraction.

[0160] (2) Preparation of detection reagents:

[0161] Table 2 lists the amount of reagent components added for each detection reaction. Due to the large number of components contained, it is necessary to prepare mixed components such as primers, probes, and basic PCR systems in advance. It can be prepared according to the amoun...

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Abstract

The invention discloses a target sequence complementary quenching probe and a kit for detecting beta globin gene point mutation. The kit comprises an asymmetric polymerase chain reaction (PCR) single-stranded deoxyribonucleic acid (DNA) template enrichment PCR amplification primer, the target sequence complementary quenching probe, LA DNA polymerase, and the like. A beta-thalassemia point mutation genotype of a tested sample can be detected by using an asymmetrical specific PCR single-stranded DNA template to enrich a human beta-globin gene mutation site target sequence and using a specific fluorescent probe to perform molecular hybridization and melting analysis. The kit has good sensitivity and accuracy for detecting wild type and mutant type of a beta-thalassemia mutation site, is excellent in repeatability and stability, and is completely suitable for the clinical detection of beta-thalassemia point mutation.

Description

technical field [0001] The invention belongs to the field of molecular detection, and in particular relates to a target sequence complementary quenching probe for detecting point mutations of beta globin genes and a kit thereof. Background technique [0002] The human β-globin gene cluster is located on chromosome 11, expresses β-globin chains, and combines with α-globin chains to form functional hemoglobin tetramers. Under normal circumstances, the ratio of α- and β-globin chains expressed by the human globin gene is appropriate (1:1). When the β-globin gene is defective, the synthesis of β-chains is reduced and the α-chains are relatively excess, which can lead to β- Thalassemia disease. Thalassemia (referred to as "thalassemia") is one of the most common single-gene genetic diseases that have the greatest impact on human health in the world. It is mainly concentrated in countries along the Mediterranean Sea, Southeast Asia, a few African regions, and southern my country....

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q2600/156
Inventor 周万军徐湘民熊符张强温晓君
Owner SOUTHERN MEDICAL UNIVERSITY
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