Jansen R W, Siegl G, Lemon S M
Department of Medicine, University of North Carolina, Chapel Hill 27599-7030.
Proc Natl Acad Sci U S A. 1990 Apr;87(8):2867-71. doi: 10.1073/pnas.87.8.2867.
We describe an immunoaffinity-linked nucleic acid amplification system (antigen-capture/polymerase chain reaction, or AC/PCR) for detection of viruses in clinical specimens and its application to the study of the molecular epidemiology of a picornavirus, hepatitis A virus (HAV). Immunoaffinity capture of virus, synthesis of viral cDNA, and amplification of cDNA by a polymerase chain reaction (PCR) were carried out sequentially in a single reaction vessel. This approach simplified sample preparation and enhanced the specificity of conventional PCR. AC/PCR detected less than one cell culture infectious unit of virus in 80 microliters of sample. Sequencing of AC/PCR reaction products from 34 virus strains demonstrated remarkable conservation at the nucleotide level among most strains but revealed hitherto unsuspected genetic diversity among human isolates. Epidemiologically related strains were identical or closely related in sequence. Virus strains recovered from epidemics of hepatitis A in the United States and Germany were identical in sequence, providing evidence for a previously unrecognized epidemiologic link between these outbreaks.
我们描述了一种用于检测临床标本中病毒的免疫亲和连接核酸扩增系统（抗原捕获/聚合酶链反应，即AC/PCR）及其在微小核糖核酸病毒甲型肝炎病毒（HAV）分子流行病学研究中的应用。在单个反应容器中依次进行病毒的免疫亲和捕获、病毒cDNA的合成以及通过聚合酶链反应（PCR）对cDNA的扩增。这种方法简化了样品制备并提高了传统PCR的特异性。AC/PCR在80微升样品中可检测到少于一个细胞培养感染单位的病毒。对34株病毒的AC/PCR反应产物进行测序表明，大多数菌株在核苷酸水平上具有显著的保守性，但揭示了人类分离株中迄今未被怀疑的遗传多样性。流行病学相关的菌株在序列上相同或密切相关。从美国和德国甲型肝炎疫情中分离出的病毒株在序列上相同，为这些疫情之间先前未被认识到的流行病学联系提供了证据。
