title: Sensitive detection of nucleic acids by PNA hybridization directed co-localization of fluorescent beads.
abstract: We have designed a pair of biotinylated peptide nucleic acid (PNA) probes targeting two sequences in 18S rRNA (from the parasite Trypanosoma brucei) at a distance of 191 nt (corresponding to maximum distance of ca. 60 nm) from each other. The PNA probes were individually bound to (strept)avidin-coated fluorescent beads, differing in size and color [green beads (1 µm) and red beads (5.9 µm)], thereby allowing distinct detection of each PNA probe by conventional fluorescence microscopy. These two PNA beads showed easily detectable co-localization when simultaneously hybridizing to a target nucleic acid. The assay detected the parasite 18S rRNA down to 1.6 fmol while there was no such co-localization visible with human 18S rRNA not containing the PNA targets. Furthermore, the assay showed positive detection with 1.6 ng of total RNA (corresponding to RNA from ca. 300 parasites). Upon further optimization this method may provide a new tool for a diagnosis of Human African Trypanosomiasis (HAT) and it may more generally have applications within diagnostics for (neglected) infectious diseases.
authors.affiliation: Department of Cellular and Molecular Medicine; Faculty of Health Sciences; The Panum Institute; University of Copenhagen; Copenhagen N, Denmark.
authors.forename: Takehiko
authors.initials: T
authors.lastname: Shiraishi
authors.forename: Stijn
authors.initials: S
authors.lastname: Deborggraeve
authors.forename: Philippe
authors.initials: P
authors.lastname: Büscher
authors.forename: Peter E
authors.initials: PE
authors.lastname: Nielsen