Hepatitis B virus enrichment fluorescent PCR (polymerase chain reaction) detection method

A hepatitis B virus and detection method technology, applied in the field of molecular biology, can solve the problems of long PCR detection time, high nucleic acid purity requirements, and high automation cost, and achieve the effects of improving detection sensitivity, short detection time, and easy operation.

Pending Publication Date: 2022-03-18
杭州丹威生物科技有限公司
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  • Description
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Problems solved by technology

[0005] The technical problem to be solved in the present invention is to overcome the deficiencies in the prior art, provide a method for detecting hepatitis B virus DNA, and solve the problems of cumbersome sample processing, high requirements for nucleic acid purity, and partial PCR detection time in the existing detection technology. Disadvantages such as long length and high automation cost

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  • Hepatitis B virus enrichment fluorescent PCR (polymerase chain reaction) detection method
  • Hepatitis B virus enrichment fluorescent PCR (polymerase chain reaction) detection method
  • Hepatitis B virus enrichment fluorescent PCR (polymerase chain reaction) detection method

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Embodiment 1

[0039] A kind of hepatitis B virus enrichment fluorescent PCR detection method, the process flow chart of this method is as follows figure 1 As shown, the specific steps are:

[0040] 1. Preparation of immunomagnetic beads

[0041] (1) activation

[0042] Take 10 mg (25 mg / mL) fully shaken carboxy magnetic beads and place them in a 2 mL centrifuge tube, add 1 ml of 15 mMMES buffer (pH6.0) to wash the magnetic beads for 3 times; after separating with a magnetic separator, add 100 μL EDC solution (prepared with 15mM MES buffer solution (pH6.0)) with a concentration of 10mg / ml was mixed by a vortex mixer, fixed on the mixer, and activated at room temperature for 30min; the magnetic beads were separated by a magnetic separator, and discarded. Remove the supernatant.

[0043] (2) Coupling

[0044] Add 400 μg hepatitis B virus surface antibody, mix and resuspend the magnetic beads, place the centrifuge tube on a horizontal shaker, and mix at room temperature for 3 hours (2-4 hou...

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Abstract

The invention relates to a hepatitis B virus enrichment fluorescent PCR (polymerase chain reaction) detection method which comprises the following steps: S1, preparation of immunomagnetic beads: coupling hepatitis B virus antibodies to carboxyl modified superparamagnetic beads to obtain the immunomagnetic beads coupled with the hepatitis B virus antibodies, s2, enrichment of hepatitis B virus: mixing and incubating hepatitis B virus positive serum or plasma with the immunomagnetic beads coupled with the hepatitis B virus antibody; s3, separating the immunomagnetic bead-virus compound by using a magnetic tool, resuspending the immunomagnetic bead-virus compound in a salt ion buffer solution, and heating and cracking; and S4, separating the magnetic beads by using a magnetic tool to obtain enriched and concentrated hepatitis B virus, and directly detecting liquid in a split product by using a fluorescent PCR reagent. According to the invention, the detection sensitivity is greatly improved due to enrichment and concentration, the specificity is strong due to immune recognition and gene recognition, and the fluorescent PCR detection time is short due to the use of a rapid PCR reagent.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for detecting hepatitis B virus enrichment fluorescent PCR. Background technique [0002] Hepatitis B Virus (HBV) is the pathogen of hepatitis B virus (referred to as hepatitis B), which is transmitted through blood and body fluids. It is an infectious disease with a chronic carrier state and mainly causes liver damage. HBV infection is one of the serious public health problems worldwide. HBV infection is prevalent worldwide. According to WHO reports, about 2 billion people worldwide have been infected with HBV, of which 350 million are chronic HBV infected, and about 1 million people die each year from liver failure, liver cirrhosis and primary disease caused by HBV infection. Hepatocellular carcinoma (hepatocellular rcinoma, HCC). There are about 100 million HBV carriers in my country, which seriously endanger the health of Chinese people and affect the quality of li...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/532G01N33/569C12Q1/686C12Q1/70C12N15/11
CPCG01N33/54326G01N33/532G01N33/56983C12Q1/686C12Q1/706G01N2333/02C12Q2563/107
Inventor 陈文孙艳刘松林胡丹枫朱小娟
Owner 杭州丹威生物科技有限公司
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