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Nested asymmetric PCR reagent kit for detecting alpha 2 globin gene point mutation

A globin and kit technology, which is applied in the field of nested asymmetric PCR melt-linking fluorescence analysis detection kits for α2 globin gene point mutation, can solve the problems of cumbersome operation, time-consuming and labor-intensive, and small detection throughput.

Active Publication Date: 2014-07-02
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the development of detection technology, amplification hindered mutation system PCR (ARMS-PCR) uses two PCR reaction tubes to detect each mutation site one by one. The operation is cumbersome, time-consuming and laborious, and the detection conditions of this technology Difficult to control, prone to false negative and false positive results
The subsequent allele-specific oligonucleotide hybridization method (ASO) greatly reduces the false negative and false positive results, but it is still necessary to detect each mutation site one by one, the detection throughput is low, time-consuming and laborious
The reverse dot blot method (RDB), which is currently routinely used in clinical practice, can detect three mutation types of WS, QS and CS at the same time, which reduces labor intensity to a certain extent and increases the detection throughput. The operation process is still cumbersome. First PCR amplifies the α2 globin gene, and then observes the test results through a series of operations such as denaturation, hybridization, membrane washing, and color development. risk of contamination
DNA sequencing analysis is also currently an optional solution for the detection of point mutations in the α-globin gene. The technical operation process is first PCR amplification, followed by PCR product purification, sequencing PCR reactions, capillary electrophoresis analysis on the sequencer, etc., which also has cumbersome operations. , carrying pollution and other issues
In general, the current detection methods for α2 globin gene point mutations are costly, heavy workload, cumbersome operation, small detection throughput, difficult to achieve automation and standardization, and there are technical limitations such as PCR product carryover contamination, which cannot meet the requirements The current large-scale population screening and clinical routine molecular diagnosis of α-thalassemia needs

Method used

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  • Nested asymmetric PCR reagent kit for detecting alpha 2 globin gene point mutation
  • Nested asymmetric PCR reagent kit for detecting alpha 2 globin gene point mutation
  • Nested asymmetric PCR reagent kit for detecting alpha 2 globin gene point mutation

Examples

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Effect test

Embodiment 1

[0076] Nested asymmetric PCR kit for detecting point mutations of α2 globin genes WS, QS, CS, CD30, CD31

[0077] 1. Kit composition

[0078] (1) A pair of primers that specifically amplify the full sequence of the α2 globin gene

[0079] F: 5'-gctcgcgggccggcact-3' (SEQ NO. 1),

[0080] R: 5'-ctccattgttggcacattccgggatag-3' (SEQ NO. 2);

[0081] (2) Two DNA single-stranded primers for amplifying the α2 globin gene sequence

[0082] The single-stranded primer sequence for amplifying the target fragment of CD30 / CD31 is: 5'-ctgcggggagaagcagagt-3' (SEQ NO.3),

[0083] The single-stranded primer sequence for amplifying the WS / QS / CS target fragment is: 5'-gggcaggaggaacggctac-3' (SEQ NO.4);

[0084] (3) Probe

[0085] Fluorescent probes that specifically detect CD30 / CD31 wild-type and CD31 (AGG>AAG) mutant types:

[0086] 5'-Cy5-ccctggagaggtgaggctaaa-3' (SEQ NO. 5);

[0087] Quencher probes that specifically detect CD30 / CD31 site wild-type and CD31 (AGG>AAG):

[0088] 5'-aatggcg...

Embodiment 2

[0130] Using the kit and method of Example 1 to detect point mutations of α-thalassemia globin genes WS, QS, CS, CD30, and CD31:

[0131] 1. Sample source and type:

[0132] Select gDNA specimens of confirmed α-thalassaemia from the sample bank of our laboratory, and the genotypes are α CD30 α / αα, α CD30 α / α CD30 α, α CD31 α / αα, α CD31 α / α CD31 1 each of α, α CS α / αα, α CS α / α CS α, α WS α / αα, α WS α / α WS α, α QS α / αα, α QS α / α QS α, -- SEA / α WS α, -- SEA / α QS α, -- SEA / α CS 2 copies of α, αα / αα each, dilute gDNA samples to 100-200ng / μl with sterilized double distilled water for later use.

[0133] 2. Sample detection:

[0134] Apply the above-mentioned reaction system of this kit (Table 1) to the above-mentioned gDNA specimen to be tested, and use the PCR reaction program of the present invention (pre-denaturation at 95°C for 8 minutes; 95°C for 30sec+72°C for 60sec, 30 cycles; 95°C for 30sec+ 63°C 20sec, 40 cycles; 72°C extension for 5min; 95°C denat...

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Abstract

The invention discloses a nested asymmetric PCR reagent kit for detecting alpha 2 globin gene point mutation. The reagent kit comprises a nested asymmetric PCR amplification primer, a fluorescent probe, a quenching probe, an LADNA polymerase and the like. The alpha-thalassemia point mutation genotype of a detected sample is detected by virtue of specific amplification of five point mutation destination fragments WS, QS, CS, CD30 and CD31 of the human alpha 2 globin gene and molecular hybridization and unwinding analysis of a specific fluorescent probe. The reagent kit has good sensitivity and accuracy to wild type and mutation type of alpha-thalassemia five point mutation positions WS, QS, CS, CD30 and CD31, has good repeatability and stability, and is totally suitable for clinical detection of alpha-thalassemia point mutation.

Description

technical field [0001] The invention belongs to the field of molecular detection, and in particular relates to a nested asymmetric PCR melting fluorescence analysis kit for detecting α2 globin gene point mutations. Background technique [0002] The human α-globin gene cluster is located on chromosome 16, expresses α-globin chains, and combines with β-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 α-globin gene is defective, the synthesis of α-chain is reduced and the β-chain is relatively excess, which can lead to α- 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. 200 million peop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6848C12Q1/6858C12Q1/6883C12Q2600/156C12Q2527/107C12Q2531/107C12Q2549/119
Inventor 周万军熊符徐湘民
Owner SOUTHERN MEDICAL UNIVERSITY
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