Quantitative PCR detection method for hepatic tissue HBVcccDNA

A detection method and liver tissue technology are applied in the field of HBVcccDNA quantitative PCR detection of liver tissue to achieve the effects of improving specificity, improving sensitivity and increasing target sequences

Inactive Publication Date: 2016-07-13
陈延平
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Problems solved by technology

So far, there is no recognized, relatively stable test kit for detecting HBVcccDNA that has been officially approved by relevant department

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  • Quantitative PCR detection method for hepatic tissue HBVcccDNA
  • Quantitative PCR detection method for hepatic tissue HBVcccDNA

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Embodiment Construction

[0035] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

[0036] Such as figure 1 Shown, liver tissue HBVcccDNA quantitative PCR detection method comprises the following steps,

[0037]Step S1: Extract the genomic DNA sample from the liver tissue, specifically grind the liver tissue, transfer it into a 1.5ml EP tube, add 200μl buffer GA, shake until completely suspended; add 20μl proteinase K solution to the above suspension and mix well to obtain The first mixed solution, wherein the mixing condition is 56°C, 3h; c...

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Abstract

The invention relates to a quantitative PCR detection method for hepatic tissue HBVcccDNA. The method comprises the following steps: extracting a genome DNA specimen in hepatic tissues; establishing a beta-actin plasmid standard; taking total RNA of adult peripheral blood cells as a template, performing inverse transcription to synthesize cDNA; performing PCR amplification on corresponding cDNA target fragments of human beta-actin genes by taking the cDNA as a template, establishing to form recombinant plasmid by taking pMD18-T as a carrier, performing electrophoresis and sequencing identification, and making a beta-actin standard curve by using fluorescent quantitative PCR. According to the method disclosed by the invention, HBVcccDNA and beta-actin in the hepatic tissues can be subjected to sensitive and specific amplification, quantification of the HBVcccDNA in hepatic cells is realized, and the method can be used for detection and application of HBVcccDNA in the clinical hepatic tissue and hepatic cells.

Description

technical field [0001] The main technical method of the present invention is real-time fluorescence quantitative PCR (Quantitativereal-timePCR, qRT-PCR), the basic principle of this technology is based on three items of molecular hybridization, gene amplification, and photoelectricity, adding fluorescent groups in the PCR reaction system, The process of PCR reaction is monitored in real time with the accumulation of fluorescent signal along with the PCR reaction, and the technology of detecting and analyzing the PCR reaction through analysis software. Background technique [0002] In the 1880s, Miller et al. used DNA electrophoresis and Southern hybridization detection techniques to first confirm that there was an HBV DNA molecule with an electrophoretic band of 2.0 kb in the liver tissue of patients with chronic hepatitis B, and it was confirmed under an electron microscope that it was about 3.2 kb in size. Supercoiled DNA molecule, the first discovery and report of the exi...

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6848C12Q1/6851C12Q2531/113C12Q2545/101C12Q2545/113C12Q2563/107
Inventor 陈延平李春艳刘志刚
Owner 陈延平
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