title: Evaluation of a microfluidic chip system for preparation of bacterial DNA from swabs, air, and surface water samples.
abstract: The detection of bacterial pathogens from complex sample matrices by PCR requires efficient DNA extraction. In this study, a protocol for extraction and purification of DNA from swabs, air, and water samples using a microfluidic chip system was established. The optimized protocol includes a combination of thermal, chemical and enzymatic lysis followed by chip-based DNA purification using magnetic particles. The procedure was tested using Gram-positive Bacillus thuringiensis Berliner var. kurstaki as a model organism for Bacillus anthracis and the attenuated live vaccine strain of Francisella tularensis subsp. holarctica as Gram-negative bacterium. The detection limits corresponded to 103genome equivalents per milliliter (GE/ml) for surface water samples spiked with F. tularensis and 102GE/ml for B. thuringiensis. In air, 10 GE of F. tularensis per 10 L and 1 GE of B. thuringiensis per 10 L were detectable. For swab samples obtained from artificially contaminated surfaces the detection limits were 4 × 103GE/cm2for F. tularensis and 4 × 102GE/cm2for B. thuringiensis. Suitability of the chip-assisted procedure for DNA preparation of real samples was demonstrated using livestock samples. The presence of thermophilic Campylobacter spp. DNA could be confirmed in air samples collected on pig and broiler farms.
authors: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Bacterial Infections and Zoonoses, Naumburger Straße 96a, 07743 Jena, Germany.
authors: Sandra
authors: S
authors: Julich
authors: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Bacterial Infections and Zoonoses, Naumburger Straße 96a, 07743 Jena, Germany.
authors: Helmut
authors: H
authors: Hotzel
authors: microfluidic ChipShop, Stockholmer Straße 20, 07747 Jena, Germany.
authors: Claudia
authors: C
authors: Gärtner
authors: Bertin Technologies, 10 Avenue André Marie Ampére, 78180 Montigny-le-Bretonneux, France.
authors: Daniel
authors: D
authors: Trouchet
authors: Mansoura University, Faculty of Veterinary Medicine, Department of Animal Hygiene and Zoonoses, 60 El Gomhoria Street, 35516 Mansoura, Egypt.
authors: Marwa
authors: M
authors: Fawzy El Metwaly Ahmed
authors: University of Veterinary Medicine Hannover, Foundation, Institute of Animal Hygiene, Animal Welfare and Farm Animal Behaviour, Bischofsholer Damm 15, 30173 Hannover, Germany.
authors: Nicole
authors: N
authors: Kemper
authors: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Bacterial Infections and Zoonoses, Naumburger Straße 96a, 07743 Jena, Germany. Electronic address: herbert.tomaso@fli.bund.de.
authors: Herbert
authors: H
authors: Tomaso
mesh_terms: D000391:Air Microbiology; D001419:Bacteria / Q000737:chemistry*; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification*; D056656:Lab-On-A-Chip Devices; D014871:Water Microbiology
chemical_list: D004269:DNA, Bacterial
keywords: Bacillus thuringiensis; Campylobacter; DNA preparation; Environmental samples; Francisella tularensis; Microfluidic chips