title: Laser capture microdissection of bacterial cells targeted by fluorescence in situ hybridization.
abstract: Direct cultivation-independent sequence retrieval of unidentified bacteria from histological tissue sections has been limited by the difficulty of selectively isolating specific bacteria from a complex environment. Here, a new DNA isolation approach is presented for prokaryotic cells. By this method, a potentially pathogenic strain of the genus Brachyspira from formalin-fixed human colonic biopsies were visualized by fluorescence in situ hybridization (FISH) with a 16S rRNA-targeting oligonucleotide probe, followed by laser capture microdissection (LCM) of the targeted cells. Direct 16S rRNA gene PCR was performed from the dissected microcolonies, and the subsequent DNA sequence analysis identified the dissected bacterial cells as belonging to the Brachyspira aalborgi cluster 1. The advantage of this technique is the ability to combine the histological recognition of the specific bacteria within the tissue with molecular analysis of 16S rRNA gene or other genes of interest. This method is widely applicable for the identification of noncultivable bacteria and their gene pool from formalin-fixed paraffin-embedded tissue samples.
authors.affiliation: Danish Institute for Food and Veterinary Research, Copenhagen, Denmark.
authors.forename: Kirstine
authors.initials: K
authors.lastname: Klitgaard
authors.forename: Lars
authors.initials: L
authors.lastname: Mølbak
authors.forename: Tim K
authors.initials: TK
authors.lastname: Jensen
authors.forename: Christian Fredrik
authors.initials: CF
authors.lastname: Lindboe
authors.forename: Mette
authors.initials: M
authors.lastname: Boye
mesh_terms: D017400:Brachyspira / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis*; D004275:DNA, Ribosomal / Q000032:analysis; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007834:Lasers; D042282:Microdissection / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S