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Fluorescent quantitative PCR (Polymerase Chain Reaction) testing method for cccDNA (covalently closed circular deoxyribonucleic acid) of hepatitis B virus and kit thereof

A hepatitis B virus, fluorescent quantitative technology, applied in the direction of fluorescence/phosphorescence, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problem of incomplete positive strands, chimeric primers or initial probes that cannot complement it Combination and other issues

Inactive Publication Date: 2012-01-25
SHANGHAI XINGYAO MED TECH DEV CO LTD +1
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  • Abstract
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Problems solved by technology

What's more, some scholars designed semi-nested PCR to detect cccDNA in PBMC, which is equivalent to amplifying the probability of this non-specific amplification by tens of thousands of times! The main basis for distinguishing rcDNA from cccDNA by chimeric primers and invasion assays is that the positive strand of rcDNA is incomplete, so chimeric primers or initial probes cannot complement it.

Method used

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  • Fluorescent quantitative PCR (Polymerase Chain Reaction) testing method for cccDNA (covalently closed circular deoxyribonucleic acid) of hepatitis B virus and kit thereof
  • Fluorescent quantitative PCR (Polymerase Chain Reaction) testing method for cccDNA (covalently closed circular deoxyribonucleic acid) of hepatitis B virus and kit thereof

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Experimental program
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Embodiment 1

[0025] Embodiment 1——The method and kit thereof for the detection of hepatitis B virus cccDNA fluorescent quantitative PCR

[0026] 1. Composition of cccDNA fluorescent quantitative PCR diagnostic kit for hepatitis B virus:

[0027] A pair of specific primers PHBV, a TaqMan fluorescent probe FPHBV, Taq enzyme, Plasmid-Safe ATP-dependent DNase enzyme, hepatitis B virus cccDNA positive control, negative control, hepatitis B virus cccDNA quantitative standard and PCR Buffer.

[0028] 2. According to the full sequence of HBV, in the front C coding region of the HBV genome, use Beacon Designer2.1 software to design and synthesize primers PHBV-S, PHBV-AS and probe FPHBV with the following sequences:

[0029] Primer PHBV-S:

[0030] 5'-TCCCCGTCTGTGCCTTC-3'(nt1549-nt1565)

[0031] Primer PHBV-AS:

[0032] 5'-CCCCAAAGCCACCCAA-3'(nt 1904-nt 1889)

[0033] Probe FPHBV:

[0034] 5'-FAM-ATCTGCCGGACCGTGTGC-TAMMRA-3'(nt1567-nt1582)

[0035] 3. Preparation of reaction solution: Each rea...

Embodiment 2

[0040] Embodiment 2——clinical testing

[0041] 150 cases of positive hepatitis B clinical samples were detected by the above method, among which 141 cases of HBVcccDNA were detected, accounting for 94% of the total, and the accurate quantitative analysis of HBVcccDNA was far superior to the general method. The invention has the advantages of good specificity, high sensitivity, accurate quantification, rapidity and convenience, and good repeatability. The invention designs primers spanning two gaps, so that rcDNA cannot be amplified, while cccDNA can be selectively amplified. In addition, the PSAD enzyme is used to process the extracted sample DNA, which can effectively reduce the non-cccDNA amplification and further improve the sensitivity and specificity of detection. The invention can not only be used for the detection of HBVcccDNA, but also can be used as an auxiliary diagnosis method for HBV infection in a clinical laboratory and a monitoring means for clinical treatment ...

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Abstract

The invention relates to a fluorescent quantitative PCR testing method for the cccDNA of the hepatitis B virus and a kit thereof. Firstly, the complete genomic sequence of the hepatitis B virus of the human body is retrieved, a pair of primers and a TaqMvan fluorescent dye-marked probe are designed for the conserved sequence of the hepatitis B virus, and the real-time fluorescent quantitative PCR method is adopted to amplify the target gene. The invention adopts Plasmid-Safe ATP-dependent Dnase (PSBD) enzyme to carry out restriction digestion on the DNA sample, thus increasing the specificity and sensitivity of tests.

Description

Technical field: [0001] The invention relates to a method for detecting hepatitis B virus covalently closed circular deoxyribonucleic acid (HBVcccDNA) and the composition of a kit. Background technique: [0002] Hepatitis B virus (HBV) belongs to the hepadnavirus family. Its genome is a double-stranded relaxed circular DNA (relaxed circular, rcDNA) with a relative molecular weight of (116-210)×109 and a total length of about 312kb. In the life cycle of HBV, the formation of covalently closed circular DNA (cccDNA) is a key step. It is the original replication template of HBV and is of great significance to the replication of HBV and the establishment of infection status. . The half-life of cccDNA is about 33 to 70 days. As long as there is cccDNA in liver cells, the replication of hepatitis B virus will not stop. Even under the strong inhibition of drugs, cccDNA in liver cells is still replicating in a small amount. , It is common to see recurrence of the patient's conditio...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 张继明江培学赵旭杨志军陈伟华吴大治夏懿
Owner SHANGHAI XINGYAO MED TECH DEV CO LTD
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