Alpha and beta thalassemia point mutation screening method

Abstract

The invention discloses an alpha and beta thalassemia point mutation screening method, and aims to provide an alpha and beta thalassemia point mutation screening method having the advantages of simplicity, rapidness, low cost and high accuracy. The method is characterized in that the method comprises the following steps: 1, extracting the genome DNA of a specimen; 2, mixing PCR mix with template DNA, and adding an LC Green Plus saturated dye; 3, carrying out PCR amplification; and 4, directly putting a product obtained after the PCR ending into an HRM analyzer, denaturing the PCR amplification product at 60-96DEG C, acquiring dense fluorescent signal changes through the optical detection system of the analyzer and drawing a temperature melting curve in the denaturing period, and accurately and automatically sorting wild and heterozygous mutation and homozygous mutation according to the curve by software.

Description

technical field [0001] The invention relates to a screening method, in particular to a screening method for common point mutations of α and β thalassemia. Background technique [0002] Currently, common screening techniques for thalassemia include: hemoglobin electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), etc. These screening techniques are mainly biochemical analysis of hemoglobin components, all of which are at the non-molecular level, and the coincidence rate of them with genetic diagnosis cannot reach 100%, which may cause missed diagnosis in clinical practice. Except that capillary electrophoresis can be used to screen HbCS mutations (1), there is no effective screening method for non-deletion α-thalassemia mutations (especially HbQS, HbWS) at home and abroad, which often leads to non-deletion HbH mutations in clinical practice. Missed diagnosis of disease. [0003] Genetic diagnosis methods for thalassemia mainly include: g...

AI Technical Summary

Problems solved by technology

RFLP performs enzyme digestion reaction by identifying specific sequence sites. The method is simple and low-cost, but its limitation is that it can only detect limited mutations that can produce enzyme cleavage sites. Due to incomplete or partial enzyme cleavage reactions, lead to false positive or false negative results(2)

ARMS-PCR needs to design corresponding primers for each mutation, and the amplification conditions of each pair of primers need to be optimized. If multiple mutations need to be detected at the same time, the operation is cumbersome, and false positive or false negative results may also occur (3)

The RDB method can detect multiple point mutations at the same time. Domestic commercial kits can detect 17 mutations of β-thalassaemia and 3 mutations of non-deletion α-thalassemia, but the operation time of this method is relatively long (about 8 hours). cumbersome

These two detection technologies for common mutation types in specific populations can achieve diagnostic accuracy and timeliness, but they cannot detect new mutations

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