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Biochem Biophys Res Commun. 2008 Oct 31;375(4):522-5. doi: 10.1016/j.bbrc.2008.08.082. Epub 2008 Aug 26.

Rapid detection and identification of a pathogen's DNA using Phi29 DNA polymerase.

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  • 1Department of Medicine, T-16 Room 027, State University of New York at Stony Brook, Stony Brook, NY 11794-8154, USA. jbruno@notes.cc.sunysb.edu

Abstract

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.

PMID:
18755142
[PubMed - indexed for MEDLINE]
PMCID:
PMC2900840
Free PMC Article

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