title: Enhanced detection of pathogenic enteric viruses in coastal marine environment by concentration using methacrylate monolithic chromatographic supports paired with quantitative PCR.
abstract: Currently, around 50% of the world's population lives in towns and cities within 100 km of the coast. Monitoring of viruses that are frequently present in contaminated coastal environments, such as rotavirus (RoV) and norovirus (NoV), which are also the major cause of human viral gastroenteritis, is essential to ensure the safe use of these water bodies. Since exposure to as few as 10-100 particles of RoV or NoV may induce gastrointestinal disease, there is a need to develop a rapid and sensitive diagnostic method for their detection in coastal water samples. In this study, we evaluate the application of methacrylate monolithic chromatographic columns, commercially available as convective interaction media (CIM®), to concentrate pathogenic enteric viruses from saline water samples prior to virus quantification by one-step reverse transcription quantitative PCR (RT-qPCR). Using RoV and NoV as model enteric viruses, we present our results on the most effective viral concentration conditions from saline water matrices using butyl (C4) hydrophobic interaction monolithic support (CIM®C4). C4 monolithic columns exhibit a good capacity to bind both RoV and NoV and both viruses can be eluted in a single step. Our protocol using a 1 ml C4 column enables processing of 400 ml saline water samples in less than 60 min and increases the sensitivity of RoV and NoV detection by approximately 50-fold and 10-fold respectively. The protocol was also scaled up using larger capacity 8 ml C4 columns to process 4000 ml of seawater samples with concentration factors of 300-fold for RoV and 40-fold for NoV, without any significant increase in processing time. Furthermore, C4 monolithic columns were adapted for field use in an on-site application of RoV concentration from seawater samples with performance equivalent to that of the reference laboratory setup. Overall, the results from successful deployment of CIM C4 columns for concentration of rotavirus and norovirus in seawater samples reiterate the utility of monolithic supports as efficient, scalable and modular preparative tools for processing environmental water samples to enhance viral detection using molecular methods.
authors.affiliation: BioSistemika LLC, Koprska 98, SI-1000, Ljubljana, Slovenia. Electronic address: bmukundh@gmail.com.
authors.forename: Mukundh N
authors.initials: MN
authors.lastname: Balasubramanian
authors.affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Večna Pot 111, SI-1000, Ljubljana, Slovenia. Electronic address: nejc.racki@sandoz.com.
authors.forename: Nejc
authors.initials: N
authors.lastname: Rački
authors.affiliation: Marine Biology Station, National Institute of Biology, Fornače 41, SI-6330, Piran, Slovenia. Electronic address: jose.goncalves@nib.si.
authors.forename: José
authors.initials: J
authors.lastname: Gonçalves
authors.affiliation: BioSistemika LLC, Koprska 98, SI-1000, Ljubljana, Slovenia. Electronic address: kkovac@biosistemika.com.
authors.forename: Katarina
authors.initials: K
authors.lastname: Kovač
authors.affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Večna Pot 111, SI-1000, Ljubljana, Slovenia. Electronic address: magda.tusek.znidaric@nib.si.
authors.forename: Magda T
authors.initials: MT
authors.lastname: Žnidarič
authors.affiliation: Marine Biology Station, National Institute of Biology, Fornače 41, SI-6330, Piran, Slovenia. Electronic address: valentina.turk@nib.si.
authors.forename: Valentina
authors.initials: V
authors.lastname: Turk
authors.affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Večna Pot 111, SI-1000, Ljubljana, Slovenia. Electronic address: maja.ravnikar@nib.si.
authors.forename: Maja
authors.initials: M
authors.lastname: Ravnikar
authors.affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Večna Pot 111, SI-1000, Ljubljana, Slovenia. Electronic address: ion.gutierrez@nib.si.
authors.forename: Ion
authors.initials: I
authors.lastname: Gutiérrez-Aguirre
mesh_terms: D002845:Chromatography; D006801:Humans; D008689:Methacrylates / Q000737:chemistry; D029322:Norovirus / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014780:Viruses
chemical_list: D008689:Methacrylates
keywords: Fecal contamination; Norovirus; Rotavirus; Seawater; Sewage; qPCR