Biological article for detecting hepatitis b virus covalence closed DNA and use thereof
A hepatitis B virus and biological technology, applied in the field of biological products for detecting hepatitis B virus covalently closed DNA, can solve the problems of low sensitivity, complicated operation and high reagent cost, and achieve the effects of strong specificity and high sensitivity
Inactive Publication Date: 2009-04-01
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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Problems solved by technology
The earliest detection of cccDNA was mostly Southern blot qualitative detection, which was limited to the field of scientific research due to its low sensitivity, cumbersome operation, and high reagent cost, which greatly limited its application value.
In recent years, there have been many reports on the detection of cccDNA by PCR technology. However, due to the high sensitivity of PCR technology, the sequences of rcDNA and cccDNA are highly homologous. Therefore, when using PCR technology to detect cccDNA, it is necessary to ensure that only amplified cccDNA is amplified, but rcDNA is not amplified
Due to the above-mentioned deficiencies of the existing techniques for detecting cccDNA, there is no stable, reliable and sensitive biological product for detecting cccDNA at present.
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[0016] The present invention uses the characteristics of the fluorescent dye EvaGreen combined with double-stranded DNA to establish a new HBV cccDNA fluorescent quantitative PCR detection system. According to the structural differences between rcDNA and cccDNA, the system designs primers spanning two gaps to prevent rcDNA from being amplified. , in order to further avoid the amplification caused by the high amount of template, the cccDNA samples extracted from the serum were treated with Plasmid-Safe ATP-dependent DNase, which can act on single-stranded and double-stranded DNA with gaps, while for intact The covalently closed DNA has no effect, and the rcDNA content in the treated sample is less, which reduces the non-specific pairing of primers. There is thus provided the biological product of the present invention.
[0017] The biological product provided by the invention is a kit for detecting hepatitis B virus HBV cccDNA based on fluorescent quantitative PCR technology. ...
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The invention discloses a biological supply used for detecting hepatitis B virus covalently-closed DNA, which comprises specific PCR primers spanning two nicks of a positive chain and a negative chain of an HBV genome, cccDNA extracting solution and treating fluid, and quantitative standards; the specific PCR primers are HBV-ccc-F 5'- CGGTCTGGGGCAAAGCTCAT-3' and 5'-GCACAGCTTGGAGGCTTGA-3'which aim at the upstreams of the nicks of the positive chain and the negative chain of the hepatitis B virus genome; and the length of the amplication fragments of the two primers is 550 bases. The biological supply is a kit which can detect cccDNA with the copy number of less than 100, is beneficial for learning about the infectivity of hepatitis B patients, and provides a reference index for the diagnosis, the observation of curing and curative effect, and the screening of hepatitis B virus resistant drugs of clinical HBV infection.
Description
technical field [0001] The invention relates to a biological product for detecting hepatitis B virus covalently closed DNA (cccDNA) and its application. Background technique [0002] HBV covalently closed circular DNA (HBV cccDNA) is the original template for HBV pregenomic RNA replication. During the replication process of hepatitis B virus, the viral DNA enters the host cell nucleus, and under the action of DNA polymerase, the gaps in the two strands are filled to form a supercoiled covalent, closed, circular DNA molecule (cccDNA) , this covalently closed DNA molecule is of great significance to the replication of hepatitis B virus and the establishment of an infectious state. cccDNA can not only be used as an index to evaluate the condition and infectivity of hepatitis B patients, but also can be used as a new index to evaluate anti-hepatitis B virus drugs. The earliest detection of cccDNA was mostly Southern blot qualitative detection, which was limited to the field of...
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Login to View More IPC IPC(8): C12Q1/70C12Q1/68
Inventor 许君尹飞飞王华林邓菲胡志红
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI