title: Development and evaluation of an ELIME assay to reveal the presence of Salmonella in irrigation water: Comparison with Real-Time PCR and the Standard Culture Method.
abstract: A reliable, low-cost and easy-to-use ELIME (Enzyme-Linked-Immuno-Magnetic-Electrochemical) assay for detection of Salmonella enterica in irrigation water is presented. Magnetic beads (MBs), coupled to a strip of eight-magnetized screen-printed electrodes localized at the bottom of eight wells (8-well/SPE strip), effectively supported a sandwich immunological chain. Enzymatic by-product is quickly measured by chronoamperometry, using a portable instrument. With the goal of developing a method able to detect a wide range of Salmonella serotypes, including S. Napoli and S. Thompson strains responsible for various community alerts, different kinds of MBs, antibodies and blocking agents were tested. The final system employs MBs coated with a broad reactivity monoclonal antibody anti-salmonella and blocked with dry milk. For a simple and rapid assay these two steps were performed in a preliminary phase, while the two sequential incubations for the immuno-recognition events were merged in a single step of 1h. In parallel a Real-Time PCR (RTi-PCR) method, based on a specific locked nucleic acid (LNA) fluorescent probe and an internal amplification control (IAC), was carried out. The selectivity of the ELIME and RTi-PCR assays was proved by inclusivity and exclusivity tests performed analyzing different Salmonella serotypes and non-target microorganisms, most commonly isolated from environmental sources. Furthermore, both methods were applied to experimentally and not experimentally contaminated irrigation water samples. Results confirmed by the ISO culture method, demonstrated the effectiveness of ELIME and RTi-PCR assays to detect a low number of salmonella cells (1-10 CFU/L) reducing drastically the long analysis time usually required to reveal this pathogen.
authors.affiliation: Department of Chemical Science and Technologies, "Tor Vergata" University, Via della Ricerca Scientifica, Rome, Italy. Electronic address: giulia.volpe@uniroma2.it.
authors.forename: G
authors.initials: G
authors.lastname: Volpe
authors.affiliation: Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome, Italy. Electronic address: elisabetta.delibato@iss.it.
authors.forename: E
authors.initials: E
authors.lastname: Delibato
authors.affiliation: Department of Chemical Science and Technologies, "Tor Vergata" University, Via della Ricerca Scientifica, Rome, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Fabiani
authors.affiliation: Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome, Italy.
authors.forename: E
authors.initials: E
authors.lastname: Pucci
authors.affiliation: Department of Chemical Science and Technologies, "Tor Vergata" University, Via della Ricerca Scientifica, Rome, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Piermarini
authors.affiliation: Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome, Italy.
authors.forename: A
authors.initials: A
authors.lastname: D'Angelo
authors.affiliation: Department of Food Inspection, Istituto Zooprofilattico Sperimentale del Mezzogiorno, Via della Salute 2, Portici, NA, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Capuano
authors.affiliation: Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome, Italy.
authors.forename: D
authors.initials: D
authors.lastname: De Medici
authors.affiliation: Department of Chemical Science and Technologies, "Tor Vergata" University, Via della Ricerca Scientifica, Rome, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Palleschi
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D001681:Biological Assay; D046508:Culture Techniques; D055664:Electrochemical Techniques; D005456:Fluorescent Dyes; D005618:Fresh Water / Q000382:microbiology; D018189:Immunomagnetic Separation; D009841:Oligonucleotides; D060888:Real-Time Polymerase Chain Reaction; D012475:Salmonella / Q000276:immunology / Q000302:isolation & purification*; D014873:Water Pollutants / Q000302:isolation & purification*
chemical_list: D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D014873:Water Pollutants; C477371:locked nucleic acid
keywords: ELIME assay; Irrigation water; RTi-PCR; S. Napoli; S. Thompson