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

StephenW

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.

StephenW

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


††现住址：Celltrion公司，学院-RO 51，延寿区，仁川，406-840南 - 韩国
生物学技术，卷。 59，5号，2015年十一月，第287-293
全文（PDF）
抽象的

包壳，其中前基因组RNA（pgRNA）被包装到病毒核衣壳后，乙型肝炎病毒（HBV）使用pgRNA作为模板通过反转录复制其DNA基因组。迄今为止，只有两个壳体化的检测方法，用于评估pgRNA的打包成核衣壳的量：（i）所述RNA酶保护测定法和（ii）天然琼脂糖凝胶电泳检测。然而，这些方法是复杂的和费力的，因为它们需要多次pgRNA纯化步骤之后检测通过同位素标记的探针。而且，无论测定是不适合评估以剂量依赖的方式大量的抗病毒剂。为了克服这些限制，我们使用涂覆有抗HBV核心（的HBc）抗体，通常用于酶联免疫吸附测定核衣壳捕获板，以固定的病毒核衣壳设计了一种新型的HBV壳体化测定法在96孔板形式。病毒pgRNA然后通过定量RT-PCR（RT-qPCR的）检测。这种策略允许多个病毒RNA样品的快速，方便，和定量分析，以评估衣壳化抑制剂。此外，我们的协议是可能适合用于靶向的HBV pgRNA壳体化的化合物的高通量筛选（HTS）。

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