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Method for performing two-color fluorescence polymerase chain reaction (PCR) detection on hepatitis B virus covalently closed circular (ccc) DNA, and application of kit

A hepatitis B virus, two-color fluorescence technology, applied in fluorescence/phosphorescence, microbial-based methods, biochemical equipment and methods, etc., can solve problems such as difficult quantification and interference detection results

Inactive Publication Date: 2011-02-09
BEIJING SUOAO BIOMEDTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, the detection of HBV cccDNA is mainly the method of common PCR or nested PCR, but it is difficult to quantify
However, the HBV cccDNA quantitative detection method has many problems to be solved in the detection technology, such as the need to further improve the sensitivity of the detection so that the HBV cccDNA in the blood can be accurately detected; further improve the specificity of the detection to avoid simultaneous detection of loose circular Double-stranded HBV DNA (RcDNA) interferes with test results, etc.

Method used

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  • Method for performing two-color fluorescence polymerase chain reaction (PCR) detection on hepatitis B virus covalently closed circular (ccc) DNA, and application of kit
  • Method for performing two-color fluorescence polymerase chain reaction (PCR) detection on hepatitis B virus covalently closed circular (ccc) DNA, and application of kit
  • Method for performing two-color fluorescence polymerase chain reaction (PCR) detection on hepatitis B virus covalently closed circular (ccc) DNA, and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Specimen collection and delivery

[0049] The specimen types of this kit include whole blood and liver tissue, etc. Whole blood can be collected with a sterile syringe to extract 2-3 ml of venous blood from the subject, inject it into a sterile 5 ml sodium citrate anticoagulant tube, invert it repeatedly 3-5 times to fully mix, and store it for testing. For the liver tissue, about 100-500mg of the subject’s liver tissue needs to be taken under aseptic conditions, transferred to a clean 1.5ml centrifuge tube, and stored for testing. Specimens after the above treatment can be stored at -20°C, but not more than 6 months, and can be stored at -70°C for a long time. When sending for inspection, it needs to be stored in a 0°C ice bottle.

Embodiment 2

[0050] Example 2: DNA Extraction

[0051]In order to carry out the method of the present invention, DNA extraction is required. For peripheral blood, first shake the anticoagulated blood, take 500μl and transfer it to a clean 1.5ml centrifuge tube, numbered and marked. Add 1ml 1× red blood cell lysate, shake vigorously for 30s, centrifuge at 3000g for 5min, and discard the supernatant. Repeat twice until there is no obvious red precipitate. If there is still a red precipitate, it is necessary to wash the cell pellet again. Then add 1ml of normal saline, shake vigorously for 15s, centrifuge at 10000g for 5min, and discard the supernatant. Add 50 μl of nucleic acid extraction solution to the precipitate, bathe in 100°C water for 10 minutes, and centrifuge at 13,000 g for 3 minutes. The supernatant is the extracted DNA solution. For liver tissue, it is necessary to wash the blood on the surface of the liver tissue with normal saline, take about 50 mg of tissue, add 50 μl of n...

Embodiment 3

[0052] Embodiment 3: Two-color fluorescent PCR amplification detects HBV cccDNA

[0053] Take out the two-color fluorescent PCR MIX and Taq enzyme system, melt at room temperature, shake and mix well, and then centrifuge at 10,000rpm for 10s. Each test reaction system was prepared as follows:

[0054] Reagent

[0055] Calculate the amount of each reagent used, add it to a clean 0.2ml centrifuge tube of appropriate volume, mix well, centrifuge at 10,000rpm for 10s, add 26μl of the system to the set PCR reaction tube, and then add the processed sample to each tube Or positive quality control or negative quality control 4μl. Before adding the samples, the positive quality control substances were pre-diluted 10 times, 100 times, and 1000 times, and the original solution of the positive quality control substances was marked as 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 copies / ml. Centrifuge briefly at 10,000 rpm for 30 seconds after adding the sample. Put each reaction tube...

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Abstract

The invention provides a method for detecting hepatitis B virus covalently-closed circular DNA (HBV ccc DNA) from the samples of peripheral blood, hepatic tissues and the like by utilizing two-color fluorescence polymerase chain reaction (PCR) technology, namely a PCR-two-color fluorescence probe method, and a kit therefor. The method comprises the following steps of: (1) collecting the samples; (2) extracting the DNA; (3) detecting the samples by a two-color fluorescence quantitative PCR in-vitro amplification method; and (4) analyzing the samples according to the fluorescence intensity of the amplification reaction of the corresponding samples after the amplification reaction is finished, thereby judging the existence of the HBV ccc DNA in the collected samples and accurately quantifying the HBV ccc DNA by using the standard curve of a positive quality control material to realize the real-time, fast and quantitative detection of the HBV ccc DNA.

Description

1. Technical field [0001] The invention relates to a detection method of hepatitis B virus cccDNA, in particular to a method for rapidly and accurately detecting hepatitis B virus cccDNA in blood and liver tissue of a patient by using a two-color fluorescence quantitative PCR technique. The present invention further relates to a kit for detecting hepatitis B virus cccDNA. 2. Background technology [0002] Hepatitis B is an infectious disease caused by hepatitis B virus (HBV, hepatitis B virus) infection. According to relevant WHO statistics, there are more than 200 million asymptomatic hepatitis B virus carriers worldwide. In my country, the infection rate of hepatitis B virus is about 60%. Asymptomatic hepatitis B virus carriers account for about 7% of the total population, and one-third of them have clinical manifestations of liver damage. At present, there are 30 million hepatitis B patients in my country. The onset of hepatitis B is characterized by slow onset, and sub...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/93C12Q1/70G01N21/64
Inventor 史成军徐贵峰任美峰刘健翊
Owner BEIJING SUOAO BIOMEDTECH
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