title: Hydrophobic ionic liquids for quantitative bacterial cell lysis with subsequent DNA quantification.
abstract: DNA is one of the most frequently analyzed molecules in the life sciences. In this article we describe a simple and fast protocol for quantitative DNA isolation from bacteria based on hydrophobic ionic liquid supported cell lysis at elevated temperatures (120-150 °C) for subsequent PCR-based analysis. From a set of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide was identified as the most suitable for quantitative cell lysis and DNA extraction because of limited quantitative PCR inhibition by the aqueous eluate as well as no detectable DNA uptake. The newly developed method was able to efficiently lyse Gram-negative bacterial cells, whereas Gram-positive cells were protected by their thick cell wall. The performance of the final protocol resulted in quantitative DNA extraction efficiencies for Gram-negative bacteria similar to those obtained with a commercial kit, whereas the number of handling steps, and especially the time required, was dramatically reduced. Graphical Abstract After careful evaluation of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide ([BMPyr+][Ntf2-]) was identified as the most suitable ionic liquid for quantitative cell lysis and DNA extraction. When used for Gram-negative bacteria, the protocol presented is simple and very fast and achieves DNA extraction efficiencies similar to those obtained with a commercial kit. ddH2O double-distilled water, qPCR quantitative PCR.
authors.affiliation: Christian Doppler Laboratory for Monitoring of Microbial Contaminants, University of Veterinary Medicine, Veterinärplatz 1, 1210, Vienna, Austria.
authors.forename: Sabine
authors.initials: S
authors.lastname: Fuchs-Telka
authors.affiliation: Christian Doppler Laboratory for Monitoring of Microbial Contaminants, University of Veterinary Medicine, Veterinärplatz 1, 1210, Vienna, Austria.
authors.forename: Susanne
authors.initials: S
authors.lastname: Fister
authors.affiliation: Christian Doppler Laboratory for Monitoring of Microbial Contaminants, University of Veterinary Medicine, Veterinärplatz 1, 1210, Vienna, Austria.
authors.forename: Patrick-Julian
authors.initials: PJ
authors.lastname: Mester
authors.affiliation: Institute for Milk Hygiene, Milk Technology, and Food Science, University of Veterinary Medicine, Veterinärplatz 1, 1210, Vienna, Austria.
authors.forename: Martin
authors.initials: M
authors.lastname: Wagner
authors.affiliation: Christian Doppler Laboratory for Monitoring of Microbial Contaminants, University of Veterinary Medicine, Veterinärplatz 1, 1210, Vienna, Austria. peter.rossmanith@vetmeduni.ac.at.|Institute for Milk Hygiene, Milk Technology, and Food Science, University of Veterinary Medicine, Veterinärplatz 1, 1210, Vienna, Austria. peter.rossmanith@vetmeduni.ac.at.
authors.forename: Peter
authors.initials: P
authors.lastname: Rossmanith
mesh_terms: D001419:Bacteria / Q000737:chemistry / Q000166:cytology / Q000235:genetics; D005591:Chemical Fractionation / Q000379:methods; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D057927:Hydrophobic and Hydrophilic Interactions; D007094:Imides / Q000737:chemistry*; D052578:Ionic Liquids / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods; D011759:Pyrrolidines / Q000737:chemistry*; D013696:Temperature
chemical_list: C581933:1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide; D004269:DNA, Bacterial; D007094:Imides; D052578:Ionic Liquids; D011759:Pyrrolidines
keywords: DNA extraction; Escherichia coli; Ionic liquids; Quantitative PCR; Salmonella Typhimurium; Solvent extraction; Two-phase system