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cccDNA standard substance, preparation method thereof, and method and kit for quantitatively detecting cccDNA of hepatitis b virus

A technology for quantitative detection of hepatitis B virus, applied in the field of molecular biology, can solve problems such as difficulty in obtaining cccDNA, inaccurate quantification of cccDNA, and impact on the process and results of fluorescent quantitative PCR

Active Publication Date: 2014-07-02
SHENZHEN INST OF ADVANCED TECH
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Problems solved by technology

However, due to the difficulty in obtaining cccDNA positive standards, most of them are cccDNA linear double-stranded fragments obtained by PCR amplification. influence, making the quantification of cccDNA inaccurate

Method used

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  • cccDNA standard substance, preparation method thereof, and method and kit for quantitatively detecting cccDNA of hepatitis b virus
  • cccDNA standard substance, preparation method thereof, and method and kit for quantitatively detecting cccDNA of hepatitis b virus
  • cccDNA standard substance, preparation method thereof, and method and kit for quantitatively detecting cccDNA of hepatitis b virus

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Embodiment Construction

[0036] Such as figure 1 As shown, the traditional detection of cccDNA by real-time fluorescent quantitative PCR is realized by a pair of special primers spanning the double gap of rcDNA and a fluorescent probe. The sense primer P1 is complementary to the negative strand and is located upstream of the positive strand gap. The antisense primer P2 is complementary to the positive strand and is located downstream of the negative strand gap. rcDNA is not amplified because of the gap, but cccDNA can be selectively amplified . A fluorescent probe complementary to the negative strand is added downstream of the cccDNA negative strand gap, which can realize the quantitative analysis of cccDNA by fluorescent quantitative PCR.

[0037]However, the method for detecting cccDNA by real-time fluorescent quantitative PCR has the following disadvantages: 1. When PCR detects cccDNA, due to the presence of rcDNA, the amount of initial template cannot be too high, otherwise primers P1 and P2 will...

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Abstract

The invention discloses a cccDNA standard substance and a preparation method thereof. The cccDNA standard substance comprises a unit of DNA of HBV genome and a recombination site attR. The invention also discloses a method and kit for quantitatively detecting the cccDNA of hepatitis B by using the cccDNA standard substance. The method utilizes mung-bean nuclease to eliminate the rcDNA interference, at the same time HBV mini-circle DNA is prepared as the cccDNA standard substance, and then a PCR technology is combined with a fluorescence labeled probe hybrid method so as to carry out quantitative detection on the cccDNA of hepatitis B virus. Compared to the conventional realtime fluorescent quantitative PCR detection method, the method has more precise quantification.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a cccDNA standard product and a preparation method thereof, a method for quantitatively detecting hepatitis B virus cccDNA and a kit. Background technique [0002] After hepatitis B virus (HBV virus) infects the host, the HBV virus relaxed circular DNA (rcDNA) enters the host cell nucleus to form a covalently closed circular supercoiled double-stranded DNA molecule genome, which covalently closes the circular supercoiled double strand DNA molecules can form a circular DNA structure, called covalently closed circular DNA (cccDNA). cccDNA is the replication "pool" of hepatitis B virus, and accurate quantification of cccDNA can assist in the diagnosis of hepatitis B virus. [0003] cccDNA and rcDNA are very different in structure: rcDNA has gaps or nicks on both the positive and negative strands, and only some regions are complementary to form a circular structure, but not a superh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/10C12Q1/70C12Q1/68
Inventor 陈志英何成宜王天燕
Owner SHENZHEN INST OF ADVANCED TECH
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