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

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...

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

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