title: Development of a degenerated TaqMan real-time Q-PCR for detection of bacteria-free DNA in dialysis fluid.
abstract: Bacterial-derived DNA fragments (BDNAs) have been shown to be present in a dialysis fluid, to pass through dialyzer membranes, and to induce interleukin 6 (IL-6) in mononuclear cells. DNA fragments are thought to be derived from microorganisms inhabiting hemodialysis water and fluid. The primary aim of the present study was to develop two degenerated TaqMan real-time quantitative-PCR (Q-PCR) for detection of a broad range of bacterial DNA that specifically detect 16S ribosomal DNA (rDNA) (862 and 241 bp) and evaluate the efficiency of the Bellco Selecta resin to capture the BDNAs in the dialysis fluid. For this purpose, we decided to compare measurements of unfragmented samples (9.8 × 105Escherichia coli genome) with artificially fragmented DNA samples. We assessed two broad-range real-time PCR targeting bacterial 16S rRNA genes for detection of fragmented and unfragmented bacterial DNA in the dialytic fluid and demonstrated that Bellco Selecta resin is capable of retaining these types of bacterial DNA.
authors.affiliation: Dipartimento di Scienze della Sanità Pubblica e Pediatriche, Scuola di Medicina, Università di Torino, Torino, Italy.|Laboratorio di Citoimmunodiagnostica, Azienda Ospedaliero-Universitaria Città della Salute e della Scienza di Torino, Torino, Italy.
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Bergallo
authors.affiliation: SC Nefrologia-Dialisi e Trapianto Pediatrico, Azienda Ospedaliero-Universitaria Città della Salute e della Scienza di Torino, Torino, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Loiacono
authors.affiliation: Dipartimento di Scienze della Sanità Pubblica e Pediatriche, Scuola di Medicina, Università di Torino, Torino, Italy.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Rizzo
authors.affiliation: Bellco srl, Mirandola, Italy.
authors.forename: Marialuisa
authors.initials: M
authors.lastname: Caiazzo
authors.affiliation: Bellco srl, Mirandola, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Palladino
authors.affiliation: SC Nefrologia-Dialisi e Trapianto Pediatrico, Azienda Ospedaliero-Universitaria Città della Salute e della Scienza di Torino, Torino, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Amore
mesh_terms: D004269:DNA, Bacterial / Q000235:genetics*; D004275:DNA, Ribosomal / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D020459:Genes, rRNA; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S
keywords: bacteria-free DNA; broad-range PCR; degenerated primer; dialysis; real-time PCR