长片段聚合酶链反应及其在肝炎病毒中的应用：甲型、乙型和丙型肝炎病毒基因组的扩增
Long PCR and its application to hepatitis viruses: amplification of hepatitis A, hepatitis B, and hepatitis C virus genomes.
作者信息
Tellier R, Bukh J, Emerson S U, Miller R H, Purcell R H
机构信息
Hepatitis Viruses Section, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, USA.
出版信息
J Clin Microbiol. 1996 Dec;34(12):3085-91. doi: 10.1128/jcm.34.12.3085-3091.1996.
Abstract
In this study we amplified virtually the entire genomes of hepatitis A virus (a member of the Picornaviridae family), hepatitis B virus (a member of the Hepadnaviridae family), and hepatitis C virus (a member of the Flaviviridae family) by using the recently described technique of long PCR. In order to do this, we first demonstrated, using the lambda phage, that long PCR can be made highly sensitive and the sensitivity can be further enhanced by nested long PCR. We also showed, using tobacco mosaic virus as a model, that a reverse transcriptase reaction can be linked to a long PCR, enabling the nearly full-length amplification of the genomes of RNA viruses. We then applied these techniques to serial dilutions of titrated stocks of well-characterized strains of hepatitis A, B, and C viruses. We amplified the nearly full-length sequence of each of these viruses from a small number of viral genomes, demonstrating the sensitivity of the process.
摘要
在本研究中，我们运用最近描述的长片段PCR技术，对甲型肝炎病毒（小核糖核酸病毒科成员）、乙型肝炎病毒（嗜肝DNA病毒科成员）和丙型肝炎病毒（黄病毒科成员）的几乎整个基因组进行了扩增。为此，我们首先利用λ噬菌体证明，长片段PCR可具有高度敏感性，且通过巢式长片段PCR可进一步提高其敏感性。我们还以烟草花叶病毒为模型表明，逆转录反应可与长片段PCR相连接，从而实现RNA病毒基因组的近全长扩增。然后，我们将这些技术应用于甲型、乙型和丙型肝炎病毒特征明确的标准毒株滴定原液的系列稀释液。我们从少量病毒基因组中扩增出了每种病毒的近全长序列，证明了该方法的敏感性。
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