Taqman fluorescence probe quantitative PCR method adopting plasmid constructed by tree shrew CD81 gene as standard substance

Abstract

The invention relates to a method for detecting and estimating the change situation of CD81 molecule which is infected by hepatitis C virus (HCV) in vivo and in vitro, in particular to a Taqman fluorescence probe quantitative PCR detection method on the basis of using cloned tree shrew CD81 plasmid as standard substance, belongs to the molecular biology field. The method comprises the following steps: cloning tree shrew CD81 gene segment; designing reasonable probe and primer with Primers Express2.0 software on the cloned tree shrew CD81 gene segment; designing experiment to select annealing temperature (Tm value), primer concentration and probe concentration under optimal conditions; further adopting tree shrew CD81 plasmid as standard substance to perform 10 times concentration gradient dilution and building standard curve; verifying the repeatability, stability and sensitivity of the method of the invention; and finally detecting the change of CD81 molecule which is infected by hepatitis C virus (HCV) in vitro by the method of the invention. The method can detect the change situation of tree shrew CD81 accurately, fast and conveniently.

Description

Technical field: [0001] The invention belongs to the field of molecular biology, and relates to a method for detecting and evaluating the change of CD81 molecules before and after tree shrews are infected with HCV (hepatitis C virus) in vivo and in vitro, and more specifically, relates to a cloned tree shrew CD81 The plasmid is used as a standard, and a detection method using Taqman probe fluorescent quantitative PCR is established on this basis. Background technique: [0002] Hepatitis C is caused by hepatitis C virus (HCV) infection, mainly transmitted by blood / body fluids, and accounts for 70% of post-transfusion hepatitis. HCV infection is a major cause of chronic liver disease worldwide. WHO estimates that 170 million people worldwide are infected with HCV. The anti-HCV positive rate in the healthy population in my country is 0.7% to 3.1%, about 38 million people [2]. HCV infection can easily lead to chronicity, and 50% to 80% of adults with acute infection are likel...

AI Technical Summary

Problems solved by technology

Since no suitable small animal model of HCV has been found, and the receptors and co-receptors of HCV have not been determined, the research on CD81, one of the most important receptors, is limited to the research on human CD81. Tree shrews as a It is very likely to be a suitable small animal model for HCV, and little is known about its CD81

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