title: Pathogen detection by core-shell type aptamer-magnetic preconcentration coupled to real-time PCR.
abstract: The presence of pathogenic bacteria is a major health risk factor in food samples and the commercial food supply chain is susceptible to bacterial contamination. Thus, rapid and sensitive identification methods are in demand for the food industry. Quantitative polymerase chain reaction (PCR) is one of the reliable specific methods with reasonably fast assay times. However, many constituents in food samples interfere with PCR, resulting in false results and thus hindering the usability of the method. Therefore, we aimed to develop an aptamer-based magnetic separation system as a sample preparation method for subsequent identification and quantification of the contaminant bacteria by real-time PCR. To achieve this goal, magnetic beads were prepared via suspension polymerization and grafted with glycidylmethacrylate (GMA) brushes that were modified into high quantities of amino groups. The magnetic beads were decorated with two different aptamer sequences binding specifically to Escherichia coli or Salmonella typhimurium. The results showed that even 1.0% milk inhibited PCR, but our magnetic affinity system capture of bacteria from 100% milk samples allowed accurate determination of bacterial contamination at less than 2.0 h with limit of detection around 100 CFU/mL for both bacteria in spiked-milk samples.
authors.affiliation: School of Medicine, Istanbul Kemerburgaz University, 34217 Istanbul, Turkey. Electronic address: cengiz.ozalp@kemerburgaz.edu.tr.
authors.forename: V Cengiz
authors.initials: VC
authors.lastname: Ozalp
authors.affiliation: Biochemical Processing and Biomaterial Research Laboratory, Gazi University, 06500 Teknikokullar, Ankara, Turkey; Department of Chemistry, Faculty of Sciences, Gazi University, 06500 Teknikokullar, Ankara, Turkey.
authors.forename: Gulay
authors.initials: G
authors.lastname: Bayramoglu
authors.affiliation: Department of Biology, Middle East Technical University, Ankara, Turkey; Test and Calibration Center, Turkish Standards Institute (TSE), 41400, Gebze, Kocaeli, Turkey.
authors.forename: Murat
authors.initials: M
authors.lastname: Kavruk
authors.affiliation: Department of Biology, Middle East Technical University, Ankara, Turkey.
authors.forename: Batuhan B
authors.initials: BB
authors.lastname: Keskin
authors.affiliation: Department of Biology, Middle East Technical University, Ankara, Turkey.
authors.forename: Huseyin A
authors.initials: HA
authors.lastname: Oktem
authors.affiliation: Biochemical Processing and Biomaterial Research Laboratory, Gazi University, 06500 Teknikokullar, Ankara, Turkey.
authors.forename: M Yakup
authors.initials: MY
authors.lastname: Arica
mesh_terms: D053000:Analytic Sample Preparation Methods / Q000379:methods*; D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000378:metabolism*; D019453:Escherichia coli O157 / Q000302:isolation & purification* / Q000378:metabolism; D005516:Food Microbiology; D060328:Magnetic Phenomena; D008863:Microspheres; D011108:Polymers / Q000737:chemistry; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012485:Salmonella typhi / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; D011108:Polymers
keywords: Aptamers; Biosensors; Magnetic beads; Pathogens; rtPCR