Development of a novel hepatitis B virus encapsidation detection assay by viral nucleocapsid-captured quantitative RT-PCR
Dong-Kyun Ryu†1, Yeji Ahn2, Wang-Shick Ryu2, and Marc P. Windisch1
1Hepatitis Research Laboratory, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, South-Korea
2Department of Biochemistry, Yonsei University, Seoul, South-Korea
††Present address: Celltrion Inc., Academy-ro 51, Yeonsu-gu, Incheon, 406-840 South-Korea
BioTechniques, Vol. 59, No. 5, November 2015, pp. 287–293
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Abstract
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. 作者: StephenW 时间: 2015-11-10 13:36
由病毒核壳拍摄定量RT-PCR的新型乙肝病毒壳体化检测分析的发展
董筠刘某†1，液剂Ahn2，望Shick Ryu2，和Marc P. Windisch1
1Hepatitis研究实验室，法国巴斯德研究所韩国城南市，京畿道，南韩国
生物化学，延世大学，首尔，南韩国教研室