Qualitative and absolute quantification kit for detecting hepatitis B virus cccDNA

Abstract

The invention discloses a qualitative and absolute quantification kit for detecting hepatitis B virus cccDNA. The kit comprises an HBV DNA extraction reagent, Plasmid-Safe TMATP-Dependent DNase, an upsteam primer with a DNA sequence of SEQ ID NO.1, a downstream primer with a DNA sequence of SEQ ID NO.2, and a Taqman probe with a DNA sequence of SEQ ID NO.3. The kit also comprises EvaGreen fluorescent dye, common PCR DNA polymerase and digital PCR DNA polymerase. According to the kit, the cccDNA can be rapidly subjected to qualitative and quantitative detection, and the kit can be used for clinically and rapidly judging whether cccDNA is completely eliminated after drug treatment.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a qualitative kit, an absolute quantitative kit and a method for effectively detecting hepatitis B virus DNA and hepatitis B virus covalently closed circular DNA. Background technique [0002] Polymerase chain reaction (polymerase chain reaction, referred to as PCR) is also called in vitro enzymatic gene amplification. Invented by Mullis in 1983, it is famous for its sensitive, specific and rapid nucleic acid analysis technology, which is a breakthrough in molecular biology technology. The basic principle is that it simulates the natural replication process of DNA. After the primers are complementary to the DNA template according to base pairing, under the action of DNA polymerase, according to the principle of base pairing (A, T, C, G), from the primers to Synthesis of a DNA strand complementary to the template DNA begins. After one cycle of denaturation, annealing, and extension...

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Problems solved by technology

However, the problem is that if the first-round amplification product is directly used as a template without purification, it will directly affect the efficiency of the second-round amplification due to the complexity of the components, and if it is purified, it will affect the accuracy.

[0016] At present, the detection method of cccDNA mentioned in the patent is based on the real-time quantitative PCR detection method invented by HE and others. This method is to detect cccDNA by fluorescent quantitative PCR with the primer probe designed for the negative strand gap of HBV, but in the case of this method , will also produce rcDNA products, the specificity is poor, and the fluorescent quantitative PCR technology uses the standard as a reference, it is only a relative quantitative technology, it cannot be absolutely quantitative, and the sensitivity is relatively low

In addition, the qualitative detection of cccDNA currently uses the method of Southern blot. This method is a classic method in molecular biology, but it has high technical requirements and low sensitivity, and it is not easy to popularize in clinical practice.

[0017] It can be seen that the above-mentioned quantitative methods have certain shortcomings, and there are still problems such as low sensitivity, poor specificity, high cost, time-consuming and inconvenient operation in clinical application.

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