title: Food Targeting: A Real-Time PCR Assay Targeting 16S rDNA for Direct Quantification of Alicyclobacillus spp. Spores after Aptamer-Based Enrichment.
abstract: Spore-forming Alicyclobacillus spp. are able to form metabolites that induce even in small amounts an antiseptical or medicinal off-flavor in fruit juices. Microbial contaminations could occur by endospores, which overcame the pasteurization process. The current detection method for Alicyclobacillus spp. can take up to 1 week because of microbiological enrichment. In a previous study, DNA aptamers were selected and characterized for an aptamer-driven rapid enrichment of Alicyclobacillus spp. spores from orange juice by magnetic separation. In the present work, a direct quantification assay for Alicyclobacillus spp. spores was developed to complete the two-step approach of enrichment and detection. After mechanical treatment of the spores, the isolated DNA was quantified in a real-time PCR-assay targeting 16S rDNA. The assay was evaluated by the performance requirements of the European Network of Genetically Modified Organisms Laboratories (ENGL). Hence, the presented method is applicable for direct spore detection from orange juice in connection with an enrichment step.
authors.affiliation: †Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany.
authors.forename: Tim
authors.initials: T
authors.lastname: Hünniger
authors.affiliation: †Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany.
authors.forename: Christine
authors.initials: C
authors.lastname: Felbinger
authors.affiliation: †Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany.
authors.forename: Hauke
authors.initials: H
authors.lastname: Wessels
authors.affiliation: ‡Lehrstuhl für Hygiene und Technologie der Milch, Tierärtzliche Fakultät, Ludwig-Maximilians-Universität, Schönleutnerstraße 8/219, 85764 Oberschleißheim, Germany.
authors.forename: Sophia
authors.initials: S
authors.lastname: Mast
authors.affiliation: †Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany.
authors.forename: Antonia
authors.initials: A
authors.lastname: Hoffmann
authors.affiliation: †Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Schefer
authors.affiliation: ‡Lehrstuhl für Hygiene und Technologie der Milch, Tierärtzliche Fakultät, Ludwig-Maximilians-Universität, Schönleutnerstraße 8/219, 85764 Oberschleißheim, Germany.
authors.forename: Erwin
authors.initials: E
authors.lastname: Märtlbauer
authors.affiliation: †Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany.
authors.forename: Angelika
authors.initials: A
authors.lastname: Paschke-Kratzin
authors.affiliation: †Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Fischer
mesh_terms: D056450:Alicyclobacillus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D001628:Beverages / Q000032:analysis / Q000382:microbiology*; D032084:Citrus sinensis / Q000737:chemistry / Q000382:microbiology*; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D005506:Food Contamination / Q000032:analysis*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013171:Spores, Bacterial / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S
keywords: 16S rDNA; Alicyclobacillus; detection; off-flavor; orange juice; real-time PCR; spores