Kidd A H, Jonsson M, Garwicz D, Kajon A E, Wermenbol A G, Verweij M W, De Jong J C
Department of Medical Microbiology, University of Lund, The Netherlands.
J Clin Microbiol. 1996 Mar;34(3):622-7. doi: 10.1128/jcm.34.3.622-627.1996.
In most clinical situations involving adenovirus infection, subgenus (subgroup) identification of an adenovirus isolate is as informative as a finer identification by serotype. A PCR method which allows the identification of human adenovirus isolates as members of subgenera A, B:1, B:2, C, D, E, or F is described. It is based on a simple (nonnested) PCR using primers which bind to regions immediately flanking the VA RNA-encoding regions of human adenovirus genomes. The PCR allows amplification of DNA from all 49 human adenovirus prototype strains so far described. Since there are differences in the lengths of the VA RNA-encoding regions in adenoviruses of different subgenera, it is possible to differentiate some subgenera according to the size of the PCR product determined by electrophoresis. This forms the basis of an initial broad categorization of isolates as belonging to either (i) subgenus B:1, C, D, or E or (ii) subgenus A, B:2, or F. Subgenus identification is completed by a one-step restriction enzyme digestion and gel electrophoresis. The method was assessed by blind subgenus identification of 200 miscellaneous primate adenovirus isolates prepared by the reference laboratory at Bilthoven, The Netherlands. Identification at the subgenus level by PCR correlated 91.5% with the results of serotyping. A further 5.5% of isolates were correctly identified as belonging to one of two specified subgenera. Six of the 200 identifications (3%) were unsuccessful for various reasons, including weak PCR products, intermediate strains, and mistaken primate host. The method should serve as a rapid means of confirming adenovirus cytopathic effects in laboratories performing virus culture, with simultaneous subgenus identification of the isolate. It will also have relevance as an aid to conventional serotyping for epidemiological purposes, since for all adenoviruses except those belonging to subgenus D, neutralization tests need only involve a maximum of four type-specific antisera.
在大多数涉及腺病毒感染的临床情况下，腺病毒分离株的亚属（亚组）鉴定与通过血清型进行更精细的鉴定同样具有参考价值。本文描述了一种聚合酶链反应（PCR）方法，该方法可将人腺病毒分离株鉴定为A、B:1、B:2、C、D、E或F亚属的成员。它基于一种简单（非巢式）PCR，使用与人类腺病毒基因组中VA RNA编码区域紧邻的侧翼区域结合的引物。该PCR可扩增出迄今已描述的所有49种人腺病毒原型株的DNA。由于不同亚属的腺病毒中VA RNA编码区域的长度存在差异，因此可以根据电泳确定的PCR产物大小来区分一些亚属。这构成了将分离株初步大致分类为（i）B:1、C、D或E亚属或（ii）A、B:2或F亚属的基础。亚属鉴定通过一步限制性内切酶消化和凝胶电泳完成。该方法通过对荷兰比尔瑟姆参考实验室制备的200种灵长类腺病毒杂项分离株进行盲法亚属鉴定来评估。通过PCR进行亚属水平的鉴定与血清分型结果的相关性为91.5%。另外5.5%的分离株被正确鉴定为属于两个指定亚属之一。200次鉴定中有6次（3%）由于各种原因未成功，包括PCR产物较弱、中间菌株以及灵长类宿主错误等。该方法应作为在进行病毒培养的实验室中确认腺病毒细胞病变效应的快速手段，同时对分离株进行亚属鉴定。它对于传统血清分型用于流行病学目的也将具有辅助作用，因为对于除D亚属以外的所有腺病毒，中和试验最多只需要涉及四种型特异性抗血清。
