Development of a novel hepatitis B virus encapsidation detection assay by viral nucleocapsid-captured quantitative RT-PCR
After encapsidation, where pregenomic RNA (pgRNA) is packaged into viral nucleocapsids, hepatitis B virus (HBV) uses the pgRNA as a template to replicate its DNA genome by reverse transcription. To date, there are only two encapsidation detection methods for evaluating the amount of pgRNA packaged i...
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2015-11-01
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| Series: | BioTechniques |
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| Online Access: | https://www.future-science.com/doi/10.2144/000114354 |
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| author | Dong-Kyun Ryu Yeji Ahn Wang-Shick Ryu Marc P. Windisch |
| author_facet | Dong-Kyun Ryu Yeji Ahn Wang-Shick Ryu Marc P. Windisch |
| author_sort | Dong-Kyun Ryu |
| collection | DOAJ |
| description | After encapsidation, where pregenomic RNA (pgRNA) is packaged into viral nucleocapsids, hepatitis B virus (HBV) uses the pgRNA as a template to replicate its DNA genome by reverse transcription. To date, there are only two encapsidation detection methods for evaluating the amount of pgRNA packaged into nucleocapsids: (i) the RNase protection assay and (ii) the native agarose gel electrophoresis assay. However, these methods are complex and laborious because they require multiple pgRNA purification steps followed by detection via an isotope-labeled probe. Moreover, both assays are unsuitable for evaluating a large number of antiviral agents in a dose-dependent manner. To overcome these limitations, we devised a novel HBV encapsidation assay in a 96-well plate format using nucleocapsid capture plates coated with an anti-HBV core (HBc) antibody, usually employed in enzyme-linked immunosorbent assays, to immobilize viral nucleocapsids. Viral pgRNA is then detected by quantitative RT-PCR (RT-qPCR). This strategy allows fast, convenient, and quantitative analysis of multiple viral RNA samples to evaluate encapsidation inhibitors. Furthermore, our protocol is potentially suitable for high-throughput screening (HTS) of compounds targeting HBV pgRNA encapsidation. |
| format | Article |
| id | doaj-art-bf63f10c92c34c44af9a4a84d3cf0f98 |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2015-11-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-bf63f10c92c34c44af9a4a84d3cf0f982025-08-20T02:25:50ZengTaylor & Francis GroupBioTechniques0736-62051940-98182015-11-0159528729310.2144/000114354Development of a novel hepatitis B virus encapsidation detection assay by viral nucleocapsid-captured quantitative RT-PCRDong-Kyun Ryu0Yeji Ahn1Wang-Shick Ryu2Marc P. Windisch31Hepatitis Research Laboratory, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, South-Korea2Department of Biochemistry, Yonsei University, Seoul, South-Korea2Department of Biochemistry, Yonsei University, Seoul, South-Korea1Hepatitis Research Laboratory, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, South-KoreaAfter encapsidation, where pregenomic RNA (pgRNA) is packaged into viral nucleocapsids, hepatitis B virus (HBV) uses the pgRNA as a template to replicate its DNA genome by reverse transcription. To date, there are only two encapsidation detection methods for evaluating the amount of pgRNA packaged into nucleocapsids: (i) the RNase protection assay and (ii) the native agarose gel electrophoresis assay. However, these methods are complex and laborious because they require multiple pgRNA purification steps followed by detection via an isotope-labeled probe. Moreover, both assays are unsuitable for evaluating a large number of antiviral agents in a dose-dependent manner. To overcome these limitations, we devised a novel HBV encapsidation assay in a 96-well plate format using nucleocapsid capture plates coated with an anti-HBV core (HBc) antibody, usually employed in enzyme-linked immunosorbent assays, to immobilize viral nucleocapsids. Viral pgRNA is then detected by quantitative RT-PCR (RT-qPCR). This strategy allows fast, convenient, and quantitative analysis of multiple viral RNA samples to evaluate encapsidation inhibitors. Furthermore, our protocol is potentially suitable for high-throughput screening (HTS) of compounds targeting HBV pgRNA encapsidation.https://www.future-science.com/doi/10.2144/000114354hepatitis B viruspregenomic RNAencapsidationantiviral assay |
| spellingShingle | Dong-Kyun Ryu Yeji Ahn Wang-Shick Ryu Marc P. Windisch Development of a novel hepatitis B virus encapsidation detection assay by viral nucleocapsid-captured quantitative RT-PCR BioTechniques hepatitis B virus pregenomic RNA encapsidation antiviral assay |
| title | Development of a novel hepatitis B virus encapsidation detection assay by viral nucleocapsid-captured quantitative RT-PCR |
| title_full | Development of a novel hepatitis B virus encapsidation detection assay by viral nucleocapsid-captured quantitative RT-PCR |
| title_fullStr | Development of a novel hepatitis B virus encapsidation detection assay by viral nucleocapsid-captured quantitative RT-PCR |
| title_full_unstemmed | Development of a novel hepatitis B virus encapsidation detection assay by viral nucleocapsid-captured quantitative RT-PCR |
| title_short | Development of a novel hepatitis B virus encapsidation detection assay by viral nucleocapsid-captured quantitative RT-PCR |
| title_sort | development of a novel hepatitis b virus encapsidation detection assay by viral nucleocapsid captured quantitative rt pcr |
| topic | hepatitis B virus pregenomic RNA encapsidation antiviral assay |
| url | https://www.future-science.com/doi/10.2144/000114354 |
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