title: Comparative evaluation of eleven commercial DNA extraction kits for real-time PCR detection of Bacillus anthracis spores in spiked dairy samples.
abstract: Spores of Bacillus anthracis are highly resistant and can survive conditions used for food preservation. Sample size and complexity represent the major hurdles for pathogen detection in food-related settings. Eleven commercial DNA extraction kits were evaluated for detection of B. anthracis spores by quantitative real-time PCR (qPCR) in dairy products. DNA was extracted from serial dilutions of B. anthracis spores in milk powder, cream cheese, whole milk and buttermilk. Three kits (QIAamp DNA mini kit, Invisorb Food kit I and II) were determined to produce the lowest limit of detections (LODs) with equally good performance. These kits employed lysozyme and proteinase K treatments or proteinase K in combination with cethyltrimethylamonium bromide-mediated (CTAB) precipitation of cell debris for cell disruption and DNA release. The LODs for these three kits were determined as 10(2) spores/ml of distilled water, 10(3)s pores/20 mg of powdered milk and 10(4) spores/100 mg of cream cheese, respectively. Performance testing of the QIAamp DNA mini kit demonstrated a good reproducibility and appropriate detection limits from 10(3)/ml for butter milk, 10(4)/ml for whole milk and 10(4)/100 mg for low fat cream cheese. However, DNA extraction efficiency was strongly inhibited by cream cheese with higher fat contents with an increased LOD of 10(6)/100 mg spores. This study demonstrated that qPCR detection depends directly on the appropriate DNA extraction method for an individual food matrix and bacterial agent.
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Bacterial Infections and Zoonoses, Naumburger Str. 96a, 07745 Jena, Germany. Electronic address: katja.mertens@fli.bund.de.
authors.forename: Katja
authors.initials: K
authors.lastname: Mertens
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Bacterial Infections and Zoonoses, Naumburger Str. 96a, 07745 Jena, Germany.
authors.forename: Lisa
authors.initials: L
authors.lastname: Freund
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Bacterial Infections and Zoonoses, Naumburger Str. 96a, 07745 Jena, Germany.
authors.forename: Gernot
authors.initials: G
authors.lastname: Schmoock
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Bacterial Infections and Zoonoses, Naumburger Str. 96a, 07745 Jena, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Hänsel
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Bacterial Infections and Zoonoses, Naumburger Str. 96a, 07745 Jena, Germany.
authors.forename: Falk
authors.initials: F
authors.lastname: Melzer
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Bacterial Infections and Zoonoses, Naumburger Str. 96a, 07745 Jena, Germany.
authors.forename: Mandy C
authors.initials: MC
authors.lastname: Elschner
mesh_terms: D000818:Animals; D001408:Bacillus anthracis / Q000235:genetics / Q000502:physiology*; D004269:DNA, Bacterial / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D003611:Dairy Products / Q000382:microbiology*; D005524:Food Technology / Q000379:methods* / Q000592:standards*; D005821:Genetic Techniques / Q000592:standards*; D057230:Limit of Detection; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D013171:Spores, Bacterial / Q000502:physiology; D014871:Water Microbiology
chemical_list: D004269:DNA, Bacterial
keywords: Bacillus anthracis; DNA extraction; Dairy products; Limit of detection; Quantitative real-time PCR; Spore