title: Multicenter validation study of two blockcycler- and one capillary-based real-time PCR methods for the detection of Salmonella in milk powder.
abstract: A collaborative study including 13 German laboratories was conducted to evaluate the performance of two non-patented real-time PCR methods for the detection of Salmonella in milk powder targeting the ttrC/ttrA- or the invA gene. The enrichment procedure and sample DNA preparation method prior to the real-time PCR was the same for both systems and the identical DNA extraction samples were analysed. The traditional cultural method according to EN ISO 6579:2002 for the detection of Salmonella in food was performed in each laboratory as the reference. The participants received twelve coded milk powder samples each of 25 g for the analysis. Four of them were Salmonella negative (level L0), four artificially contaminated with <3 MPN/g Salmonella Typhimurium (level L1) and four artificially contaminated with 3.6 MPN/g S. Typhimurium (level L2) to the beginning of the experiment. Of the 13 laboratories 12 used various models of real-time PCR blockcyclers conducting both real-time PCR assays and three laboratories the Light Cycler 2.0 system (Roche Bioscience) conducting the ttr-based real-time PCR assay only. The relative accuracy for both real-time PCR assays performed on blockcyclers was for level L0 97.5%. For level L1 the relative accuracy was 94.1% and for level L2 it was 100% for both assays. The relative accuracy on the Light Cycler 2.0 system was 100% for all levels applied to the ttr-real-time PCR.
authors.affiliation: Federal Institute for Risk Assessment, National Salmonella Reference Laboratory, Diedersdorfer Weg 1, D-12277 Berlin, Germany. burkhard.malorny@bfr.bund.de
authors.forename: Burkhard
authors.initials: B
authors.lastname: Malorny
authors.forename: Dietrich
authors.initials: D
authors.lastname: Mäde
authors.forename: Paul
authors.initials: P
authors.lastname: Teufel
authors.forename: Cornelia
authors.initials: C
authors.lastname: Berghof-Jäger
authors.forename: Ingrid
authors.initials: I
authors.lastname: Huber
authors.forename: Annette
authors.initials: A
authors.lastname: Anderson
authors.forename: Reiner
authors.initials: R
authors.lastname: Helmuth
mesh_terms: D000818:Animals; D001431:Bacteriological Techniques / Q000379:methods / Q000592:standards; D004269:DNA, Bacterial / Q000032:analysis*; D005506:Food Contamination / Q000032:analysis*; D006801:Humans; D008892:Milk / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012475:Salmonella / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D004269:DNA, Bacterial