A real-time fluorescent PCR method of mgb probe for detecting hla-a*31:01 allele and its combination of primers and probes

Abstract

The invention provides an MGB probe real-time fluorescent PCR (Polymerase Chain Reaction) method for detecting HLA-A*31:01 allelic genes and a primer probe composition thereof. A method for detecting the HLA-A*31:01 allelic genes is more specific by combining a method of an ARMS (Amplification Refractory Mutation System) method on the basis of using a high-specificity MGB probe detection method. The designed primer probe composition of the high-specificity amplification HLA-A*31:01 allelic genes is as follows: an upstream primer Fp:5'-GAGCCAGAGGATGGAGCC-3', a downstream primer Rp:5'-CCAGGTCCACTCGGTCtA-3', a probe probe1: 5'-FAM-aGGCCtGAGTATTGGGAC-MGB-3', and a probe probe2: 5'-VIC-CCTTCACaTTCCGTGTCTC-MGB-3', wherein FAM is 6-carboxyfluorescein, VIC is 4,7,2-trichloro-7-phenyl-6-carboxyfluorescein, and MGB is Minor Groove Binder; in addition, a primer and a probe of a reference gene are used, the specific primer and the probe of a target gene and the primer and the probe of the reference gene are added into the same tube to perform multiplex fluorescent PCR reaction; a result is analyzed through a fluorescence amplification curve. The MGB probe real-time fluorescent PCR (Polymerase Chain Reaction) method for detecting the HLA-A*31:01 allelic genes and the primer probe composition thereof have the characteristics of simplicity, convenience, flexibility, high speed, high specificity, high flux, no pollution, high sensitivity, capacity of monitoring a reaction in real time and the like, can be applied to detecting the HLA-A*31:01 allelic genes of a DNA sample of a whole genome of peripheral blood and saliva of a human body.

Description

technical field [0001] The invention belongs to the fields of pharmacogenomics and gene diagnosis, and in particular relates to a method for detecting HLA-A*31:01 alleles. Background technique [0002] Carbamazepine (CBZ) is a commonly used drug in clinical treatment of epilepsy and peripheral neuralgia. However, as one of the most common clinically induced drug eruption reactions, the use of carbamazepine also exposes patients to certain risks. This drug eruption reaction is divided into severe adverse drug reactions such as Stevens-Johnson syndrome (SJS), toxic cutaneous necrolysis (TEN), and drug reaction-associated hypereosinophilia and systemic syndrome (DRESS). And mild adverse drug reactions such as mild maculopapular eruption (MPE). The incidence rate of drug eruption reaction caused by carbamazepine is about 10%, and the incidence rate of severe drug eruption reaction such as SJS / TEN is relatively low (1-35 / ten thousand), but the fatality rate can be as high as 30...

AI Technical Summary

Problems solved by technology

The technology is simple, the technical conditions are easy to master, the sample size is small, the blood conditions are not high, and it is suitable for the clinical application of rapid detection of known HLA sequences. However, because PCR-SSP technology requires gel electrophoresis experiments, This technology is easy to cause secondary pollution and takes a long time during the operation process. At the same time, many pairs of primers in the PCR process are likely to cause non-target gene products such as dimers, which can easily cause false positive results.

These traditional HLA allele detection methods are far from meeting the growing detection needs and flexibility

Images