Xu Yun, Gao Simon, Bruno John F, Luft Benjamin J, Dunn John J
Department of Medicine, T-16 Room 027, State University of New York at Stony Brook, Stony Brook, NY 11794-8154, USA.
Biochem Biophys Res Commun. 2008 Oct 31;375(4):522-5. doi: 10.1016/j.bbrc.2008.08.082. Epub 2008 Aug 26.
Zoonotic pathogens including those transmitted by insect vectors are some of the most deadly of all infectious diseases known to mankind. A number of these agents have been further weaponized and are widely recognized as being potentially significant biothreat agents. We describe a novel method based on multiply-primed rolling circle in vitro amplification for profiling genomic DNAs to permit rapid, cultivation-free differential detection and identification of circular plasmids in infectious agents. Using Phi29 DNA polymerase and a two-step priming reaction we could reproducibly detect and characterize by DNA sequencing circular DNA from Borrelia burgdorferi B31 in DNA samples containing as little as 25 pg of Borrelia DNA amongst a vast excess of human DNA. This simple technology can ultimately be adapted as a sensitive method to detect specific DNA from both known and unknown pathogens in a wide variety of complex environments.
包括那些通过昆虫媒介传播的人畜共患病原体，是人类已知的所有传染病中最致命的一些。其中一些病原体已被进一步武器化，并被广泛认为是潜在的重大生物威胁因子。我们描述了一种基于多重引物滚环体外扩增的新方法，用于分析基因组DNA，以实现对感染因子中环状质粒的快速、无需培养的差异检测和鉴定。使用Phi29 DNA聚合酶和两步引物反应，我们能够通过DNA测序在含有低至25 pg伯氏疏螺旋体B31 DNA的DNA样本中，在大量人类DNA中可重复地检测和表征来自伯氏疏螺旋体B31的环状DNA。这种简单的技术最终可以被改编为一种灵敏的方法，用于在各种复杂环境中检测已知和未知病原体的特定DNA。
