Plastic-adherent DNA aptamer-magnetic bead and quantum dot sandwich assay for Campylobacter detection.
DNA aptamers were developed against MgCl(2)-extracted surface proteins from Campylobacter jejuni. The two highest affinity aptamers were selected for use in a magnetic bead (MB) and red quantum dot (QD)-based sandwich assay scheme. The assay was evaluated using both heat-killed and live C. jejuni and exhibits detection limits as low as an average of 2.5 colony forming unit (cfu) equivalents in buffer and 10-250 cfu in various food matrices. The assay exhibits low cross-reactivity with bacterial species outside the Campylobacter genus, but exhibits substantial cross-reactivity with C. coli and C. lari. The assay was evaluated with a spectrofluorometer and a commercially available handheld fluorometer, which yielded comparable detection limits and ranges. Remarkably, the sandwich assay components adhere to the inside face of polystyrene cuvettes even in food matrices near neutral pH, thereby enabling a rapid homogeneous assay, because fluorescence is concentrated to a small, thin planar area and background fluorescence from the bulk solution is minimized. The plastic cuvette-adherent technology coupled to a sensitive handheld fluorometer may enable rapid (15-20 min), portable detection of foodborne pathogens from "farm-to-fork" by obviating the slow enrichment culture phase used by other food safety tests.
Bruno
John G
JG
Operational Technologies Corporation, 4100 NW Loop 410, Suite 230, San Antonio, TX, 78229, USA. john.bruno@otcorp.com
Phillips
Taylor
T
Carrillo
Maria P
MP
Crowell
Randy
R
D000818:Animals; D052157:Aptamers, Nucleotide / Q000737:chemistry*; D015374:Biosensing Techniques; D002167:Campylobacter / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D003429:Cross Reactions; D005470:Fluorometry; D005516:Food Microbiology; D006801:Humans; D008280:Magnetics; D008863:Microspheres; D010865:Pilot Projects; D010969:Plastics / Q000737:chemistry*; D011137:Polystyrenes / Q000737:chemistry; D045663:Quantum Dots; D012680:Sensitivity and Specificity; D013997:Time Factors
D052157:Aptamers, Nucleotide; D010969:Plastics; D011137:Polystyrenes