Establishment of a semi-automated pathogen DNA isolation from whole blood and comparison with commercially available kits. 2011

Herbert Wiesinger-Mayr, and Elena Jordana-Lluch, and Elisa Martró, and Silvia Schoenthaler, and Christa Noehammer
AIT Austrian Institute of Technology, Molecular Medicine, A-1190 Vienna, Austria. herbert.wiesinger@ait.ac.at

Molecular methods for bacterial pathogen identification are gaining increased importance in routine clinical diagnostic laboratories. Achieving reliable results using DNA based technologies is strongly dependent on pre-analytical processes including isolation of target cells and their DNA of high quality and purity. In this study a fast and semi-automated method was established for bacterial DNA isolation from whole blood samples and compared to different commercially available kits: Looxster, MolYsis kit, SeptiFast DNA isolation method and standard EasyMAG protocol. The newly established, semi-automated method utilises the EasyMAG device combined with pre-processing steps comprising human cell lysis, centrifugation and bacterial pellet resuspension. Quality of DNA was assessed by a universal PCR targeting the 16S rRNA gene and subsequent microarray hybridisation. The DNA extractions were amplified using two different PCR-mastermixes, to allow comparison of a commercial mastermix with a guaranteed bacterial DNA free PCR mastermix. The modified semi-automated EasyMAG protocol and the Looxster kit gave the most sensitive results. After hybridisation a detection limit of 10(1) to 10(2) bacterial cells per mL whole blood was achieved depending on the isolation method and microbial species lysed. Human DNA present in the isolated DNA suspension did not interfere with PCR and did not lead to non-specific hybridisation events.

UI MeSH Term Description Entries
D001774 Blood Chemical Analysis An examination of chemicals in the blood. Analysis, Blood Chemical,Chemical Analysis, Blood,Analyses, Blood Chemical,Blood Chemical Analyses,Chemical Analyses, Blood
D004269 DNA, Bacterial Deoxyribonucleic acid that makes up the genetic material of bacteria. Bacterial DNA
D004275 DNA, Ribosomal DNA sequences encoding RIBOSOMAL RNA and the segments of DNA separating the individual ribosomal RNA genes, referred to as RIBOSOMAL SPACER DNA. Ribosomal DNA,rDNA
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D001331 Automation Controlled operation of an apparatus, process, or system by mechanical or electronic devices that take the place of human organs of observation, effort, and decision. (From Webster's Collegiate Dictionary, 1993) Automations
D001419 Bacteria One of the three domains of life (the others being Eukarya and ARCHAEA), also called Eubacteria. They are unicellular prokaryotic microorganisms which generally possess rigid cell walls, multiply by cell division, and exhibit three principal forms: round or coccal, rodlike or bacillary, and spiral or spirochetal. Bacteria can be classified by their response to OXYGEN: aerobic, anaerobic, or facultatively anaerobic; by the mode by which they obtain their energy: chemotrophy (via chemical reaction) or PHOTOTROPHY (via light reaction); for chemotrophs by their source of chemical energy: CHEMOLITHOTROPHY (from inorganic compounds) or chemoorganotrophy (from organic compounds); and by their source for CARBON; NITROGEN; etc.; HETEROTROPHY (from organic sources) or AUTOTROPHY (from CARBON DIOXIDE). They can also be classified by whether or not they stain (based on the structure of their CELL WALLS) with CRYSTAL VIOLET dye: gram-negative or gram-positive. Eubacteria
D001431 Bacteriological Techniques Techniques used in studying bacteria. Bacteriologic Technic,Bacteriologic Technics,Bacteriologic Techniques,Bacteriological Technique,Technic, Bacteriological,Technics, Bacteriological,Technique, Bacteriological,Techniques, Bacteriological,Bacteriologic Technique,Bacteriological Technic,Bacteriological Technics,Technic, Bacteriologic,Technics, Bacteriologic,Technique, Bacteriologic,Techniques, Bacteriologic
D012336 RNA, Ribosomal, 16S Constituent of 30S subunit prokaryotic ribosomes containing 1600 nucleotides and 21 proteins. 16S rRNA is involved in initiation of polypeptide synthesis. 16S Ribosomal RNA,16S rRNA,RNA, 16S Ribosomal,Ribosomal RNA, 16S,rRNA, 16S
D016133 Polymerase Chain Reaction In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain
D046228 Microarray Analysis The simultaneous analysis, on a microchip, of multiple samples or targets arranged in an array format. Microarray Analytical Devices,Microarray Microchips,Nanoarray Analytical Devices,Analysis, Microarray,Analytical Device, Microarray,Analytical Device, Nanoarray,Analytical Devices, Microarray,Analytical Devices, Nanoarray,Device, Microarray Analytical,Device, Nanoarray Analytical,Devices, Microarray Analytical,Devices, Nanoarray Analytical,Microarray Analytical Device,Microarray Microchip,Microchip, Microarray,Microchips, Microarray,Nanoarray Analytical Device

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