HBV cccDNA extraction and detection method
By using nucleocytoma separation and SYBR real-time quantitative PCR to detect cccDNA, the complexity and inaccuracy of existing methods have been solved, achieving efficient and economical detection of cccDNA and screening out effective inhibitors.
Patent Information
- Application Number
- CN202511329573.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-17
- Publication Date
- 2026-02-06
AI Technical Summary
Existing cccDNA extraction and detection methods are complex and costly, and the detection results are affected by rcDNA, resulting in inaccuracy and poor repeatability.
HBV DNA was extracted from the cytoplasm and nucleus using a cytoplasmic-nucleus separation method. SYBR real-time quantitative PCR was performed using primers that specifically recognize HBV cccDNA, and high-efficiency separation and detection were achieved using a plasmid miniprep kit.
This method enables rapid, economical, and accurate detection of cccDNA, improves the stability and reproducibility of experimental results, and allows for the screening of effective HBV cccDNA inhibitors.
Smart Images
Figure CN121472374A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioengineering technology, specifically relating to an economical, efficient, and rapid method for HBV cccDNA extraction and detection. Background Technology
[0002] Chronic hepatitis B virus (HBV) infection is a challenging global health problem. The persistent and stable presence of the virus requires the establishment and maintenance of covalently closed circular DNA (cccDNA), and the copy number of cccDNA in infected hepatocytes is lower compared to the replication intermediate (relaxed circular DNA, i.e., rcDNA).
[0003] The current classic method for cccDNA extraction is Hirt extraction, and the detection methods are Southern blotting, selective primer design combined with real-time quantitative PCR (qPCR) or digital PCR. However, when these extraction and detection methods are used together, the detection results are often affected by rcDNA, leading to inaccurate detection. Therefore, some researchers have proposed treating the contaminated rcDNA with exonuclease (T5 exonuclease) or plasmid-safe ATP-dependent DNase (PSAD). This increases the complexity and cost of experimental procedures, and the reproducibility and stability of the experimental results are questionable. Summary of the Invention
[0004] To address the shortcomings of existing technologies, the purpose of this invention is to provide an economical, rapid, and efficient real-time quantitative PCR method for extracting and detecting cccDNA using nucleoprotein-cytoplasmic separation primers. This method meets research needs while saving experimental costs and allows for simultaneous analysis of large batches of samples, making it better suited for research on new anti-HBV drugs and mechanism analysis.
[0005] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: In a first aspect, the present invention provides a method for extracting and detecting HBV cccDNA, comprising the following steps: culturing test cells, treating test cells with cell membrane lysis buffer, gently lysing the cell membrane, centrifuging, separating the cytoplasmic supernatant and the nuclear precipitate, extracting HBV DNA from the cytoplasm and the nucleus respectively using a plasmid miniprep kit, designing primers specifically for recognizing HBV cccDNA for HBV DNA in the nucleus, and performing SYBR real-time quantitative PCR detection.
[0006] In some embodiments, the primers that specifically recognize HBV cccDNA are shown in SEQ ID NO.1-2.
[0007] Furthermore, the HBV cccDNA content in the cytoplasm was detected using the primers that specifically recognize HBV cccDNA. The HBV cccDNA content in the cell nucleus was much greater than that in the cytoplasm, proving the accuracy of this method.
[0008] Secondly, the present invention provides a method for testing and / or screening HBV cccDNA inhibitors, comprising culturing an HBV-infected cell model, adding a working concentration of a candidate compound, and performing detection using the method described in the first aspect.
[0009] Thirdly, the present invention provides an HBV cccDNA inhibitor obtained by screening based on the above method, having the following structural formula: R1 is independently selected from benzoate group, p-hydroxybenzoate group, polyhydroxybenzoate group, p-methoxybenzoate group, polymethoxybenzoate group, fluorine, chlorine, or bromine substituted benzoate group, and hydrogen.
[0010] Preferably, R1 is a benzoate group or a p-hydroxybenzoate group.
[0011] Compared with the prior art, the present invention has the following beneficial effects: (1) The design concept of this invention is to achieve rapid detection of HBV cccDNA by separating the nucleus and cytoplasm and detecting the content of HBV cccDNA in the cell nucleus using real-time quantitative PCR. Experiments have shown that the detection amount of HBV cccDNA in the nucleus is much higher than that in the cytoplasm, verifying the feasibility of the above approach. Furthermore, the detection of total HBV DNA eliminates the possibility of false positives. This invention is significantly simpler and more economical than existing methods.
[0012] (2) The present invention further optimizes the kit that can efficiently separate the cytoplasm and the nucleus, which provides convenience for separating the nucleus; the use of plasmid extraction kit to assist in the extraction of HBV cccDNA ensures its high efficiency, reduces experimental time, and improves the stability and reproducibility of experimental results.
[0013] (3) Based on the above method, the present invention tests existing HBV cccDNA inhibitors and screens a class of sesquiterpene derivatives that have an inhibitory effect on HBV cccDNA. Attached Figure Description
[0014] Figure 1 Here is a flowchart of the sample preparation process; Figure 2 Flowchart for HBV cccDNA extraction and detection; Figure 3This is a schematic diagram of the experimental conditions for an RT-qPCR experiment; Figure 4 This is a diagram showing the results of HBV cccDNA detection using the nucleus-cytoplasm separation method; among them, Figure 4 In the figure, A represents the detection results of total HBV cccDNA content in the cell nucleus, cytoplasm, and cells. Figure 4 In the figure, B represents the detection results of total HBV DNA content in the cell nucleus, cytoplasm, and cells; Figure 5 A statistical graph showing the effect of cccR08 on HBV cccDNA; Figure 6 This is a statistical graph showing the effect of PAC5 on HBV cccDNA. Detailed Implementation
[0015] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.
[0016] It should be noted that any methods or instruments not specified below can be performed using conventional methods in this field. Some of the materials and their sources involved in the experiments are as follows: DMEM / F12 medium, MeilunBio, catalog number PWL005; fetal bovine serum, MeilunBio, catalog number PWL001; double antibiotics (streptomycin, penicillin), MeilunBio, catalog number MA0110; phosphate-buffered saline (PBS, pH 7.2), MeilunBio, catalog number MA0015; 0.25% trypsin (containing EDTA), MeilunBio, catalog number MA0233; SYBR-Real-Time Quantitative Mix, Novizan, catalog number Q711; Doxycycline, Maclean, catalog number D861181; nuclear / plasma protein extraction kit, Sangon Biotech, catalog number C510001; plasmid miniprep kit, Tiangen, catalog number DP103; TA cloning kit, Novizan, C601-01.
[0017] Example 1: HBV cccDNA extraction and detection method.
[0018] This embodiment provides an economical, efficient, and rapid method for HBV cccDNA extraction and detection, including the following steps: (1) Cell sample preparation As per the process Figure 1As shown, HepAD38 cells were cultured normally (90% DMEM / F12, 10% FBS, 0.3ug / ml Doxycycline) until the cell coverage reached 80%-90%. Then, they were digested with 0.25% trypsin and seeded into 6-well plates at approximately 50,000 cells / well. Doxycycline was not added to the seeding medium. One day after seeding, the test compound was added or not added, using the same medium as the seeding medium (3 ml / well). After 3 days, the medium was replaced with the compound. After 6 days, the medium was removed, and the cells were washed 2-3 times with ice-cold PBS. Cells were collected using a cell scraper, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were stored on ice.
[0019] (2) HBV cccDNA extraction As per the process Figure 2 As shown, a portion of the collected cells were processed using the C510001 kit (nuclear / plasma protein extraction kit) from Sangon Biotech Co., Ltd., following the manufacturer's instructions. Cell membranes were gently lysed, and centrifugation was performed to separate the cytoplasm (supernatant) and nuclei (precipitate). The separated cytoplasm and nuclei were then processed using the DP103 kit (plasmid mini-preparation kit) from Tiangen Biotech Co., Ltd., following the manufacturer's instructions, to extract HBV rcDNA and HBV cccDNA from the cytoplasm and nuclei, respectively. (Based on previous literature and consensus, HBV cccDNA is only present in the nucleus, while HBV rcDNA is mainly present in the cytoplasm and cell culture medium; a very small amount of HBV rcDNA present in the nucleus will eventually be modified into HBV cccDNA). Simultaneously, total HBV DNA was directly extracted from another portion of the collected cells using the DP103 kit.
[0020] (3) HBV cccDNA detection The three extracted DNA samples (HBV DNA from the cell nucleus and cytoplasm, and total HBV DNA from cells) were absolutely quantified using the primers in Table 1 via SYBR real-time quantitative PCR. The experimental system is shown in Table 2, and the experimental conditions are as follows. Figure 3 As shown, the following steps were performed sequentially: pre-denaturation (95℃, 2 minutes), PCR cycling (95℃ for 10 seconds denaturation, 60℃ for 30 seconds annealing / extension, 40 cycles), melting curve denaturation initiation (95℃, 5 seconds), setting the initial temperature (65℃ for 5 seconds), and gradient heating to 95℃.
[0021] Table 1. RT-qPCR primer sequence listing Table 2. RT-qPCR Experimental System The PCR amplification fragments (the amplification template being the HBVcccDNA extracted from the nucleus of HepAD38 cells) were ligated into the plasmid using TA cloning (Novizan C601-01, 5 min TA / Blunt-Zero Cloning Kit) with primers ZKK-cccDNA-F and ZKK-cccDNA-R. The plasmid was then transformed into E. coli DH5α competent cells. After confirming the ligation with M13F universal primers, the plasmid was extracted by shaking the culture and used as a standard for absolute quantification.
[0022] (4) Result judgment Using a nucleus-cytoplasm separation method, this invention successfully detected HBV cccDNA within the nucleus, such as... Figure 4 As shown in A, the detection amount is 10 times that detected in the cytoplasm, consistent with studies indicating the presence of HBV cccDNA in the cell nucleus. To eliminate the possibility of false positives, primers SEQ ID NO. 3-4 were designed to detect total HBV DNA (including HBV rcDNA and HBV cccDNA), as shown in... Figure 4 The results (B) showed that the total HBV DNA in the cytoplasm was more than twice that in the nucleus, further confirming the feasibility and accuracy of the HBV cccDNA detection method using the cytoplasm-nucleus separation method of this invention. Simultaneously, this invention used two pairs of primers to detect HBV cccDNA and total HBV DNA in the total number of cells (including cytoplasm and nucleus) with the same cell count. The results showed that the total HBV DNA in the total cells was two orders of magnitude higher than the HBV cccDNA. These results indicate that this experimental method can theoretically be used for HBV cccDNA detection in scientific research experiments.
[0023] Example 2: Validation of positive compounds.
[0024] An article published in the Journal of Hepatology in 2023 reported the synthesis of an HBV cccDNA inhibitor, cccR08 (DOI: 10.1016 / j.jhep.2022.12.014). This invention uses this HBV cccDNA inhibitor as a positive compound to test the feasibility of the method in Example 1. Specific sample preparation and detection are as described in Example 1. The effect of cccR08 on HBV cccDNA was detected using this method. Figure 5 As shown, the experimental results indicate that the inhibition of HBV cccDNA by cccR08 is concentration-dependent. 100 μM cccR08 showed an inhibition rate of over 90% for HBV cccDNA, while 10 μM cccR08 showed an inhibition rate of approximately 30%. This demonstrates that the experimental method of this invention can indeed detect HBV cccDNA with relatively high accuracy.
[0025] Example 3: The natural product derivative PAC5 has an inhibitory effect on HBV cccDNA.
[0026] The natural product derivative PAC5 is a small molecule compound (DOI: 10.1093 / procel / pwac027) that was previously discovered in the inventor's laboratory and has an inhibitory effect on HBV. Its structural formula is as follows: Following the extraction and detection methods described above, this invention detected a concentration-dependent inhibitory effect of PAC5 on HBV cccDNA, specifically as follows: Figure 6 As shown, 30 μM and 100 μM PAC5 inhibited HBV cccDNA by approximately 50%, while 3 μM and 10 μM PAC5 had no inhibitory effect on HBV cccDNA.
[0027] The above embodiments are used to explain the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.
Claims
1. A method for extracting and detecting HBV cccDNA, characterized in that, The procedure includes the following steps: culturing the cells to be tested, separating the cytoplasm and nucleus, extracting HBV DNA from the cytoplasm and nucleus using a plasmid miniprep kit, designing primers specifically for recognizing HBV cccDNA in the nucleus, and performing SYBR real-time quantitative PCR detection.
2. The method according to claim 1, characterized in that, The method further includes: using the primers that specifically recognize HBV cccDNA to detect the content of HBV cccDNA in the cytoplasm, in order to verify the accuracy of the method.
3. The method according to claim 1, characterized in that, The separation of cytoplasm and nucleus includes: treating the cells to be tested with cell membrane lysis buffer, gently lysing the cell membrane, centrifuging, and separating the cytoplasmic supernatant and the nucleus precipitate.
4. A method for detecting and / or screening HBV cccDNA inhibitors, characterized in that, A cell model of HBV infection was cultured, and a working concentration of the candidate compound was added. The mixture was then tested using the method described in claim 1.
5. An HBV cccDNA inhibitor screened based on the method described in claim 4, characterized in that, The inhibitor is a sesquiterpene derivative with the following structural formula: R1 is independently selected from benzoate group, p-hydroxybenzoate group, polyhydroxybenzoate group, p-methoxybenzoate group, polymethoxybenzoate group, fluorine, chlorine, or bromine substituted benzoate group, and hydrogen.
6. The HBV cccDNA inhibitor according to claim 5, characterized in that, R1 is a benzoate group or a p-hydroxybenzoate group.