title: Food Sensing: Aptamer-Based Trapping of Bacillus cereus Spores with Specific Detection via Real Time PCR in Milk.
abstract: Aerobic spores pose serious problems for both food product manufacturers and consumers. Milk is particularly at risk and thus an important issue of preventive consumer protection and quality assurance. The spore-former Bacillus cereus is a food poisoning Gram-positive pathogen which mainly produces two different types of toxins, the diarrhea inducing and the emetic toxins. Reliable and rapid analytical assays for the detection of B. cereus spores are required, which could be achieved by combining in vitro generated aptamers with highly specific molecular biological techniques. For the development of routine bioanalytical approaches, already existing aptamers with high affinity to B. cereus spores have been characterized by surface plasmon resonance (SPR) spectroscopy and fluorescence microscopy in terms of their dissociation constants and selectivity. Dissociation constants in the low nanomolar range (from 5.2 to 52.4 nM) were determined. Subsequently, the characterized aptamers were utilized for the establishment and validation of an aptamer-based trapping technique in both milk simulating buffer and milk with fat contents between 0.3 and 3.5%. Thereby, enrichment factors of up to 6-fold could be achieved. It could be observed that trapping protocol and characterized aptamers were fully adaptable to the application in milk. Due to the fact that aptamer selectivity is limited, a highly specific real time PCR assay was utilized following trapping to gain a higher degree of selectivity.
authors: Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg , Grindelallee 117, 20146 Hamburg, Germany.
authors: Christin
authors: C
authors: Fischer
authors: Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg , Grindelallee 117, 20146 Hamburg, Germany.
authors: Tim
authors: T
authors: Hünniger
authors: Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg , Grindelallee 117, 20146 Hamburg, Germany.
authors: Jan-Hinnerk
authors: JH
authors: Jarck
authors: Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg , Grindelallee 117, 20146 Hamburg, Germany.
authors: Esther
authors: E
authors: Frohnmeyer
authors: Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg , Grindelallee 117, 20146 Hamburg, Germany.
authors: Constanze
authors: C
authors: Kallinich
authors: Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg , Grindelallee 117, 20146 Hamburg, Germany.
authors: Ilka
authors: I
authors: Haase
authors: Institute of Biochemistry and Molecular Biology, University of Hamburg , Martin-Luther-King-Platz 6, 20146 Hamburg, Germany.
authors: Ulrich
authors: U
authors: Hahn
authors: Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg , Grindelallee 117, 20146 Hamburg, Germany.
authors: Markus
authors: M
authors: Fischer
mesh_terms: D000818:Animals; D001409:Bacillus cereus / Q000235:genetics / Q000302:isolation & purification*; D002417:Cattle; D005506:Food Contamination / Q000032:analysis; D008892:Milk / Q000737:chemistry / Q000382:microbiology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D052156:SELEX Aptamer Technique / Q000379:methods*; D013171:Spores, Bacterial / Q000235:genetics / Q000302:isolation & purification*
keywords: B. cereus; SPR; food poisoning; milk; real time PCR; spore trapping; spores