A detection method for hepatitis B virus based on dnazyme probe

A technology for hepatitis B virus and detection method, applied in the biological field, can solve the problems of high detection cost, unfavorable wide-scale promotion, etc., and achieve the effects of accurate detection results, true and reliable detection results, and reduction of operational complexity.

Active Publication Date: 2018-09-07
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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Problems solved by technology

[0002] At present, the most widely used detection method of hepatitis B virus DNA is real-time fluorescent quantitative PCR method. Fluorescent quantitative PCR has many advantages, including high sensitivity and strong specificity, but this technology needs to rely on expensive fluorescent quantitative PCR instruments and fluorescent probes, and the detection cost Much higher than ordinary PCR, not conducive to large-scale promotion

Method used

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  • A detection method for hepatitis B virus based on dnazyme probe
  • A detection method for hepatitis B virus based on dnazyme probe
  • A detection method for hepatitis B virus based on dnazyme probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, the qualitative detection of hepatitis B serum sample

[0036] 1. PCR reaction

[0037]

[0038]

[0039] Amplification conditions: 94°C for 2min; 94°C for 30s, 60°C for 90s, 35-40 cycles.

[0040] 2. Color reaction;

[0041] Add 400mM NaCl to the PCR product, heat at 94°C for 1min, leave at room temperature for 30min, add 2μM hemin, 2.4mM ABTS, 2mM H 2 o 2 , observe the color change with the naked eye.

[0042] 3. Test results

[0043] figure 1 It is the agarose gel electrophoresis profile of the hepatitis B virus serum sample. Among them, lanes 1, 2, and 3 are the PCR amplification products of negative hepatitis B serum samples, tested hepatitis B serum samples, and hepatitis B positive control (including the S region gene plasmid), and the size of the amplified fragment is 116bp; lane M is the molecular weight of DL-2000 Marker, the fragments are 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom. It can be seen that there is no...

Embodiment 2

[0045] Embodiment 2, the quantitative detection of hepatitis B serum sample

[0046] 1. Preparation of standard curve

[0047] The hepatitis B clinical sample that quantitatively determines the copy number of hepatitis B virus DNA is diluted with hepatitis B negative serum to form 10 1 -10 8 The copied detection standard of 8 concentration gradients was used as the test sample for amplification and detection, and three replicates were set for each sample.

[0048] (1) PCR amplification

[0049]

[0050] dNTPs1.2mM (600μM dUTP, 200μM dATP, 200μM dCTP, 200μM dGTP);

[0051]

[0052] Amplification conditions: 10min at 20°C; 2min at 95°C; 30s at 94°C, 90s at 60°C, 35-40 cycles.

[0053] (2) Colorimetric detection and drawing of standard curve

[0054] Add 400mM NaCl to the PCR product obtained in step (1), heat at 94°C for 1min, let stand at room temperature for 30min, add 2μM hemin, 2.4mM ABTS, 2mM H 2 o 2 , using a Thermo Varioskan Flash multi-function reader, read ...

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Abstract

The invention belongs to the technical field of molecular biology and discloses a method for qualitatively and quantitatively detecting hepatitis B virus DNA by using a DNAzyme probe. Aiming at the limitations of the prior art, the present invention uses PCR amplification as a detection platform, and realizes one-step qualitative and quantitative detection of hepatitis B virus by introducing a 3'-amino-modified DNAzyme probe. The technology has the advantages of high sensitivity, strong specificity, wide detection linear range, low cost, high accuracy, and convenient operation, and provides a simple, fast, accurate, efficient, economical and practical detection method for hepatitis B virus.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for qualitative and quantitative detection of hepatitis B virus DNA based on a DNAzyme probe. Background technique [0002] At present, the most widely used detection method of hepatitis B virus DNA is real-time fluorescent quantitative PCR method. Fluorescent quantitative PCR has many advantages, including high sensitivity and strong specificity, but this technology needs to rely on expensive fluorescent quantitative PCR instruments and fluorescent probes, and the detection cost Much higher than ordinary PCR, which is not conducive to large-scale promotion. Therefore, the development of a simple, rapid, accurate, efficient, economical and practical HBV detection method has important social significance and scientific research value. [0003] Deoxyribozyme (DNAzyme) is a single-stranded DNA sequence with catalytic function obtained by in vitro screening technolo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12Q1/6848C12R1/93
CPCC12Q1/6848C12Q1/686C12Q1/70C12Q2525/301C12Q2563/125C12Q2521/531
Inventor 杨丽唐卓
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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