title: Development of a fluorescent enzyme-linked DNA aptamer-magnetic bead sandwich assay and portable fluorometer for sensitive and rapid listeria detection.
abstract: A fluorescent DNA aptamer-magnetic bead sandwich assay was developed to detect listeriolysin O (LLO) protein from pathogenic Listeria bacteria using a peroxidase-linked system, Amplex Ultra Red (AUR; derivatized resazurin) substrate, and a custom-designed handheld fluorometer. The assay is highly sensitive with demonstrated limits of detection (LODs) in the range of 4 to 61 L. monocytogenes cells or the equivalent LLO produced by 4 to 61 cells on average in separate titration trials. Total assay processing and analysis time was approximately 30 mins. The assay has demonstrated the ability to detect 6 species of Listeria as desired by the USDA's Food Safety Inspection Service (FSIS). The portable system was designed to be used primarily with surface swab samples from fomites, but it can also be used to assess enrichment cultures. The minimal time to detect a positive enrichment culture in our hands from an initial 10 cell inoculum in 200 ml of broth has been 8 h post-incubation at 37 °C in shaker flask cultures. An optional automated magnetic bead assay processing and wash device capable of simultaneously processing 6 samples with low and consistent fluorescence background for higher volume central laboratories is also described.
authors.affiliation: Pronucleotein Biotechnologies Corporation, 4100 NW Loop 410, Suite 110, San Antonio, TX, 78229, USA, john.bruno@otcorp.com.
authors.forename: John G
authors.initials: JG
authors.lastname: Bruno
authors.forename: Taylor
authors.initials: T
authors.lastname: Phillips
authors.forename: Tiffany
authors.initials: T
authors.lastname: Montez
authors.forename: Adrian
authors.initials: A
authors.lastname: Garcia
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Sivils
authors.forename: Michael W
authors.initials: MW
authors.lastname: Mayo
authors.forename: Alex
authors.initials: A
authors.lastname: Greis
mesh_terms: D052157:Aptamers, Nucleotide / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000032:analysis; D001483:Base Sequence; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000379:methods*; D005470:Fluorometry / Q000295:instrumentation*; D006360:Heat-Shock Proteins / Q000032:analysis; D006460:Hemolysin Proteins / Q000032:analysis; D008089:Listeria monocytogenes / Q000302:isolation & purification*; D059346:Magnets / Q000737:chemistry*; D008863:Microspheres; D013997:Time Factors
chemical_list: D052157:Aptamers, Nucleotide; D001427:Bacterial Toxins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; C038705:hlyA protein, Listeria monocytogenes