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A method and kit for detecting and evaluating anti-HBV drug activity

A drug activity and drug technology, applied in the field of detection and evaluation of anti-HBV drug activity and kits, can solve the problems of inaccurate and comprehensive test results, achieve reasonable primer design, high gene expression level, and increased detection sensitivity Effect

Active Publication Date: 2020-06-26
北京旌准医疗科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] In view of the defects in the prior art, the object of the present invention is to provide a method and kit for detecting and evaluating the activity of anti-HBV drugs for non-therapeutic and non-diagnostic purposes, which can detect HBV pgRNA changes under the action of different drugs for use in Judging the effect of drugs to treat HBV, or screening out better anti-HBV drugs, and breaking through the shortcomings of current HBV DNA testing results that are not accurate and comprehensive

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  • A method and kit for detecting and evaluating anti-HBV drug activity
  • A method and kit for detecting and evaluating anti-HBV drug activity
  • A method and kit for detecting and evaluating anti-HBV drug activity

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Embodiment 1, real-time fluorescent quantitative PCR kit

[0049] 1. Kit composition

[0050] The kit contains: upstream primers, downstream primers, Taqman probes and reverse transcription primers; where:

[0051] The sequence of the upstream primer (F1) is:

[0052] 5'-CATGCAACTTTTTTCACCTCTGC-3' (as shown in sequence listing sequence 1);

[0053] The sequence of the downstream primer (R1) is:

[0054] 5'-GAACTGCCAATGATCGTACG-3' (as shown in sequence listing sequence 2);

[0055] The sequence of the Taqman probe (FP) is:

[0056] 5'-ATCTCATGTTCATGTCCTACTGTTCA-3' (as shown in Sequence Listing Sequence 3); its 5' end is marked with fluorescence, and its 3' end is marked with fluorescence;

[0057] The sequence of the reverse transcription primer (RT) is:

[0058] 5'-GAACTGCCAATGATCGTACGTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3' (shown in sequence 4).

[0059] The composition and content of each reagent in the kit are as follows:

[0060] Quality control: HBV pgRNA negative...

Embodiment 2

[0069] Embodiment 2, the real-time fluorescent quantitative PCR detection of HBV pgRNA content in the sample

[0070] 1. Sample extraction

[0071] Take out the HBV pgRNA internal standard quality control product, proteinase K, sedimentation aid and seven quality control products stored at -20°C, melt at room temperature, shake for 15 seconds, and centrifuge briefly.

[0072] Shake the serum to be tested for 15s and mix thoroughly. Take seven quality controls (pgRNA negative quality control, pgRNA critical positive quality control, pgRNA positive quality control and pgRNA quantitative standard (Ⅰ, Ⅱ, Ⅲ, Ⅳ)) and 200μL to 1.5mL of the serum submitted for testing In a centrifuge tube, add 300 μL of virus lysate, 5 μL of HBV pgRNA internal standard quality control, 10 μL of proteinase K, 4 μL of sedimentation aid and 20 μL of magnetic beads (mix by inversion before each pipetting), and mix gently by inversion at room temperature. Homogenize for 10 minutes to fully combine the ma...

Embodiment 3

[0093] Embodiment 3, detection experiment of anti-HBV drug activity in mice

[0094] 1. Experimental materials

[0095] (1) Main reagents: Hepatitis B drug M, FQ-PCR kit (real-time fluorescence quantitative PCR kit), HBs Ag (hepatitis B surface antigen) ELISA kit.

[0096] (2) Main instruments: automatic microplate reader, FQ real-time fluorescent quantitative PCR instrument

[0097] (3) Animals: female HBV transgenic mice, provided by the Experimental Animal Center of Peking University. The female HBV transgenic mice were developed by microinjecting 1.5 copies of HBV DNA into Balb / c mouse embryos. High levels of HBsAg and HBV DNA could be detected in the serum of the female HBV transgenic mice. In this experiment, 10-week-old adult mice were used.

[0098] 2. Experimental method

[0099] The transgenic mice were randomly divided into 3 groups, namely the positive group, the low-dose drug group and the high-dose drug group, and a blank control group (ie, female non-transg...

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Abstract

The invention relates to a method and kit for detecting and evaluating the activity of an anti-HBV drug. The method includes a step S1 of adding a to-be-tested anti-HBV drug in a culture system of animal hepatoma cells or human hepatoma cells, to perform culture; a second S2 of detecting the concentration of HBV pgRNA in culture supernatant; and a step S3 of determining activity results of the to-be-tested anti-HBV drug according to the concentration of HBV pgRNA in the culture supernatant. The kit includes an upstream primer, a downstream primer, a fluorescent labeling probe, and a reverse transcription primer, and sequences of the kit are sequences 1-4 of a sequence table. The method and the kit have the advantages of being high in detection sensitivity, sensitive, simple and convenientin the detection process and short in cycle.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method and a kit for detecting and evaluating the activity of anti-HBV drugs. Background technique [0002] Hepatitis B virus, referred to as hepatitis B, is a disease caused by hepatitis B virus HBV infection. Hepatitis B virus HBV belongs to the family Hepadnaviridae and belongs to the genus Orthohepadnavirus. The genome length is about 3.2kb, which is a partially double-stranded circular DNA. In the blood of HBV-infected patients, three different virus particles can be observed under the electron microscope at the same time: including large spherical particles with a diameter of 42nm, which are complete virus particles and are infectious, also known as Dane particles; Small spherical particles and tubular particles with a diameter of 22nm. Small spherical particles and tubular particles are hepatitis B surface antigen, without nucleic acid, and non-infectious. [...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6851
CPCC12Q1/6851C12Q2563/107
Inventor 陈广磊刘明坤叶锋
Owner 北京旌准医疗科技有限公司
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