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

A standard product, the technology of tree shrews, applied in the field of molecular biology, can solve the problems of unidentified HCV receptors and co-receptors, little knowledge about CD81, etc.

Inactive Publication Date: 2010-04-07
KUNMING UNIV OF SCI & TECH
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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 import

Method used

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

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Embodiment 1

[0044] Example 1: The establishment of the Taqman probe fluorescent quantitative PCR method using the tree shrew CD81 plasmid as a standard:

[0045] The experimental method that does not indicate specific condition in the embodiment, generally according to conventional conditions such as people such as Sambrook, molecular cloning: laboratory handbook (New York: Cold Spring Harbor Laboratory Press, 1989) or people such as Peng Xiuling, genetic engineering experiment technique, ( Science and Technology Press), or in accordance with the conditions recommended by the manufacturer. The gene map of hepatitis C virus refers to the reported literature such as Jin Qi editor-in-chief, Medical Molecular Virology (Science Press, 2001).

[0046] Cloning and sequencing of the tree shrew CD81 gene fragment:

[0047] 1. Design of PCR primers and PCR amplification

[0048] The PCR primers were designed and obtained based on more than 40 CD81 nucleic acid sequences of different species downl...

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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...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 夏雪山冯悦赵文华刘丽魏大巧
Owner KUNMING UNIV OF SCI & TECH
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