Method and kit for detecting human ATP7B gene mutation type

Abstract

The invention discloses a method and a kit for detecting a human ATP7B gene mutation type. The kit comprise a PCR amplification primer, a mass spectrum extension primer and a MassARRAY chip. The method includes the steps of firstly, preparing the PCR amplification primer; secondly, preparing the mass spectrum extension primer; thirdly, preparing a detecting template; fourthly, performing PCR amplification to obtain a target sequence amplification product; fifthly, performing SAP enzyme treatment; sixthly, using extension reaction to obtain an extension product; seventhly, purifying; eighthly, performing time of flight mass spectrometric detection. The kit and the method have the advantages that the ATP7B gene mutation sites can be simultaneously and accurately detected, and the kit and the method is high in throughput, automatic and low in cost.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to the fields of multiple PCR amplification technology and time-of-flight mass spectrometry genotyping system (MassArray). More specifically, the present invention relates to a method and kit for detecting human ATP7B gene mutant. Background technique [0002] Hepatolenticular degeneration (HLD), also known as Wilson's disease (WD), is an autosomal recessive hereditary disorder of copper metabolism. The incidence rate of different populations is about 15-30 / 1 million. Its gene is located at 13q14.3, and its cDNA encodes a P-type copper-transporting ATPase (copper-transporting P-type ATPase, ATP7B) with a molecular weight of about 165Kda consisting of 1465 amino acids. Due to the mutation of ATP7B gene in patients with WD, the ATP7B enzyme function defect located in the trans-Golgi network (TGN) of liver cells and the side of the bile duct membrane leads to the disorder of...

AI Technical Summary

Problems solved by technology

However, this method requires high personnel and equipment, and it is difficult to be popularized and applied in general hospitals; and the WD gene is about 80kb in length, and each exon has a variety of mutation forms, and it is costly and time-consuming to sequence and analyze all exons one by one , generally unbearable for patients

A large number of research results have confirmed that there is a high degree of genetic heterogeneity in the WD gene, and there are many mutation sites in each exon. At present, more than 300 forms of gene mutations have been found, and most of them are compound heterozygous mutations. It is a rare mutation, and there are obvious differences in the mutation characteristics of different races

Although this explains the complex and diverse clinical manifestations of WD patients, it brings difficulties to the clinical practice of gene diagnosis

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