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Precise quantification method of HBV (hepatitis B virus) cccDNA (covalent closed circular DNA)

A hepatitis B virus, covalent technology, applied in the field of accurate DNA quantification, can solve the problems of inaccurate cccDNA quantification, false positives, etc., and achieve the effect of simplifying the operation process and reducing the risk of cross-contamination

Inactive Publication Date: 2017-05-10
NANFANG HOSPITAL OF SOUTHERN MEDICAL UNIV
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Problems solved by technology

[0006] At present, the main problems of cccDNA quantification in human hepatocytes are: cccDNA quantification is inaccurate due to the interference of rcDNA and other forms of viruses, and the double-gap method often used in ordinary quantitative PCR instruments will be interfered by non-specific amplification of rcDNA, resulting in false positives

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  • Precise quantification method of HBV (hepatitis B virus) cccDNA (covalent closed circular DNA)
  • Precise quantification method of HBV (hepatitis B virus) cccDNA (covalent closed circular DNA)
  • Precise quantification method of HBV (hepatitis B virus) cccDNA (covalent closed circular DNA)

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[0020] Quantitative PCR across rcDNA double-gap is currently a popular cccDNA detection method. By designing primers and probes on both sides of the gap in the rcDNA double-stranded molecule, the PCR amplification reaction is terminated at the gap, thereby avoiding the amplification of the rcDNA molecule. The increase caused false positives, and because there is no gap in cccDNA, PCR can be performed normally to generate positive signals. However, in the actual reaction, there are always some rcDNA molecules whose gaps are repaired completely due to the repair characteristics of PCR amplification enzymes, and become cccDNA molecules, resulting in false positive results.

[0021] The method for the precise quantification of hepatitis B virus covalently closed circular DNA of the present invention comprises the following steps:

[0022] (1) Use a spectrophotometer to measure the OD260 and OD280 values ​​of the sample, calculate the DNA concentration and purity in the sample, and...

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Abstract

The invention relates to a precise quantification method of HBV (hepatitis B virus) cccDNA (covalent closed circular DNA). The precise quantification method of the HBV cccDNA comprises steps as follows: the copy number of a sample is quantitatively controlled within 10,000 copes through nucleic acid; primers and probes of cccDNA and rcDNA are designed and detected with a double-gap method; digital PCR (polymerase chain reaction) amplification is performed; analysis parameters are adjusted in digital PCR instrument analysis software, 1 / 2-1 / 3 of the average fluorescence intensity of positive particles in positive control holes is set as a positive threshold, the to-be-detected sample is analyzed and the copy number of cccDNA in the sample is calculated using software of the digital PCR instrument, and false positive caused by non-specific amplification is eliminated. The method solves the false positive interference problem of double-gap method and the PCR method for rcDNA; compared with other digital PCR detection methods for cccDNA, the method has the advantages that specific DNA enzyme is not required to treat the sample, closed pipe operation is realized, the cross-pollution risk is reduced, and the operation process is simplified.

Description

technical field [0001] The invention relates to a method for accurately quantifying DNA, in particular to a method for accurately quantifying hepatitis B virus covalently closed circular DNA (cccDNA). Background technique [0002] Covalently closed circular DNA (covalently closed circular DNA) is called cccDNA for short. After hepatitis B virus infects the host cell, it replicates in the cell and establishes an infection state. Enzymes etc. "repair" formed, structurally complete, supercoiled double-stranded DNA molecules. cccDNA exists in the nucleus and becomes the replication "pool" of hepatitis B virus. At present, there is no drug that can enter the nucleus and clear cccDNA. This is also one of the reasons why the existing antiviral drugs are not effective and easy to relapse after treatment. . [0003] Quantitative detection of cccDNA is difficult for hepatitis virus detection, because there are multiple homologous HBV DNA molecules (cccDNA, rcDNA, ssDNA, integrated H...

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/686C12Q2545/113C12Q2531/113C12Q2563/159
Inventor 张小勇刘红艳
Owner NANFANG HOSPITAL OF SOUTHERN MEDICAL UNIV
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