title: Novel DNA sensor system for highly sensitive and quantitative retrovirus detection using virus encoded integrase as a biomarker.
abstract: In the current study we describe a novel DNA sensor system that allows the detection of single catalytic DNA integration events mediated by retrovirus encoded integrase (IN) present in viral particles. This is achieved by rolling circle amplification mediated conversion of enzymatic reactions happening within nanometer dimensions to directly detectable micrometer sized DNA products. The system utilizes the unique integration reaction of IN to generate a surface anchored nicked DNA circle that serves as a substrate for rolling circle amplification and allows for specific, quantitative and sensitive detection of purified recombinant IN or virus particles with a detection limit of less than 30 virus particles per μL of sample. Moreover, by modifying the nucleotide sequences of the utilized DNA it was possible to tailor the system to distinguish between the highly pathogenic lentivirus HIV and the gammaretrovirus murine leukemia virus present in a given sample. Infections with HIV remain a major threat to global health with more than 2 million new infections and 1 million deaths each year. The sensitive and specific detection of HIV particles based on IN activity holds promise for the development of a new type of diagnostic tools suitable for early (within hours of infection) detection of HIV, which would be valuable for prevention strategies as well as for efficient treatment.
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark. fsp@mbg.au.dk brk@mbg.au.dk.
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.forename: Jiangnan
authors.initials: J
authors.lastname: Liu
authors.forename: Jonas
authors.initials: J
authors.lastname: Thomsen
authors.forename: Denis
authors.initials: D
authors.lastname: Selnihhin
authors.forename: Marianne S
authors.initials: MS
authors.lastname: Hede
authors.forename: Freja C M
authors.initials: FC
authors.lastname: Kirsebom
authors.forename: Oskar
authors.initials: O
authors.lastname: Franch
authors.forename: Søren
authors.initials: S
authors.lastname: Fjelstrup
authors.forename: Magnus
authors.initials: M
authors.lastname: Stougaard
authors.forename: Yi-Ping
authors.initials: YP
authors.lastname: Ho
authors.forename: Finn Skou
authors.initials: FS
authors.lastname: Pedersen
authors.forename: Birgitta R
authors.initials: BR
authors.lastname: Knudsen
mesh_terms: D001483:Base Sequence; D015374:Biosensing Techniques; D004247:DNA / Q000737:chemistry*; D057809:HEK293 Cells; D015497:HIV-1 / Q000302:isolation & purification*; D006801:Humans; D019426:Integrases / Q000737:chemistry*; D009052:Leukemia Virus, Murine / Q000302:isolation & purification*
chemical_list: D004247:DNA; D019426:Integrases