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Method for detecting specificity of Epstein-Barr viruses through Epstein-Barr virus p18-p23 fused capsid antigen

A technology for detection of EB virus and antigen, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of cumbersome steps, unsuitable for clinical application, inability to distinguish EBV latent infection and proliferative infection, etc., and achieve simple operation and reliable detection results , Ease of expressing immunogenic effects

Inactive Publication Date: 2012-03-21
邱清芳
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Problems solved by technology

Both virus isolation and molecular biology methods require certain equipment and professional personnel to operate, the steps are cumbersome, and are not suitable for clinical application, and some methods cannot distinguish EBV latent infection from proliferative infection. Serology is still the most clinically used method. detection

Method used

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Embodiment Construction

[0015] Below in conjunction with embodiment further illustrate the present invention.

[0016] Preparation of p23 and p18 fusion gene

[0017] (1) Preparation of template DNA: the target gene was obtained by PCR using the B95-8 strain virus DNA as a template. The B95-8 cells were harvested by conventional culture, and the cell DNA was extracted by the method of phenol, chloroform, and isoamyl alcohol. The steps are as follows:

[0018] Resuspend the cells with an appropriate amount of STE, add proteinase K to a final concentration of 200 μg / mL, digest at 42°C for 3-5 hours, and shake several times during this period. Add an equal volume of saturated phenol, mix upside down for 20 minutes, and centrifuge at 12000 r / min for 15 minutes at 4°C. Transfer the upper aqueous phase to a new centrifuge tube, add an equal volume of chloroform:isoamyl alcohol (24:1) for extraction, and centrifuge at 12000r / min for 5min. Transfer the upper aqueous phase to a new centrifuge tube, add 2 t...

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Abstract

The invention discloses a method for detecting specificity of Epstein-Barr viruses (EBV) through an Epstein-Barr virus p18-p23 fused capsid antigen. The method is characterized in that an overlap extension polymerase chain reaction (PCR) technology is adopted; a coding gene p23 and a coding gene p18 are fused in vitro by a coding sequence of an intermediate head (Gly4Ser)3; a fused protein is expressed and the expressed fused protein is purified and is subjected to specificity identification through an immunoblotting technology; the purified fused protein is coated on an enzyme-linked immuno sorbent assay (ELISA) reaction plate and component content and reaction conditions are optimized; commercial horseradish peroxidase-labelled goat anti-human IgM and IgG and a series of other reagents are prepared; and EBV-viral cuspid antigen (VCA) specific IgM and IgG indirect ELISA diagnostic kit is obtained by assembling.

Description

technical field [0001] The invention relates to a specific detection method for EBV. In particular, it relates to a method for detecting EBV specificity by fusion capsid antigen of Epstein-Barr virus p18 and p23. Background technique [0002] Epstein-Barr virus (EBV) is a B lymphocyte-tropic human herpes virus and is the pathogen of infectious mononucleosis (IM). closely related. EBV has the characteristics of latent infection. Under certain conditions, the latent virus can be activated, causing proliferative infection, causing multi-system lesions after infection, and the clinical manifestations are also diverse, which is easy to cause misdiagnosis and is extremely harmful to human health. Therefore, the detection of EBV infection plays an important role in diagnosing the disease and effectively monitoring the curative effect and epidemiological investigation. [0003] The current methods for diagnosing EBV infection include three aspects: ① Isolate the virus by B cell t...

Claims

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Application Information

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IPC IPC(8): G01N33/569
Inventor 邱清芳
Owner 邱清芳
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