title: Comparison of Five Commercial Nucleic Acid Extraction Kits for the PCR-based Detection ofBurkholderia PseudomalleiDNA in Formalin-Fixed, Paraffin-Embedded Tissues.
abstract: The extraction and further processing of nucleic acids (NA) from formalin-fixed paraffin-embedded (FFPE) tissues for microbiological diagnostic polymerase chain reaction (PCR) approaches is challenging. Here, we assessed the effects of five different commercially available nucleic acid extraction kits on the results of real-time PCR. FFPE samples from organs ofBurkholderia pseudomallei-infected Swiss mice were subjected to processing with five different extraction kits from QIAGEN (FFPE DNA Tissue Kit, EZ1 DNA Tissue Kit, DNA Mini Kit, DNA Blood Mini Kit, and FlexiGene DNA Kit) in combination with three different real-time PCRs targetingB. pseudomallei-specific sequences of varying length after 16 years of storage. The EZ1 DNA Tissue Kit and the DNA Mini Kit scored best regarding the numbers of successful PCR reactions. In case of positive PCR, differences regarding the cycle-threshold (Ct) values were marginal. The impact of the applied extraction kits on the reliability of PCR from FFPE material seems to be low. Interfering factors like the quality of the dewaxing procedure or the sample age appear more important than the selection of specialized FFPE kits.
authors.affiliation: Chemical Faculty, University of Hamburg , Hamburg, Germany.
authors.forename: Sonja
authors.initials: S
authors.lastname: Obersteller
authors.affiliation: Friedrich Loeffler Institute, Federal Research Institute for Animal Health , Jena, Germany.
authors.forename: Heinrich
authors.initials: H
authors.lastname: Neubauer
authors.affiliation: NATO Center of Excellence for Military Medicine (MilMedCOE), Deployment Health Surveillance Capability (DHSC) , Munich, Germany.
authors.forename: Ralf Matthias
authors.initials: RM
authors.lastname: Hagen
authors.affiliation: Department of Tropical Medicine at the Bernhard Nocht Institute, German Armed Forces Hospital of Hamburg, Hamburg, Germany; Institut for Medical Microbiology, Virology and Hygiene, University Medicine Rostock, Rostock, Germany.
authors.forename: Hagen
authors.initials: H
authors.lastname: Frickmann
keywords: Burkholderia pseudomallei; PCR; formalin-fixed; nucleic acid extraction; paraffin-embedded; tissue