title: Evaluation of Two Triplex One-Step qRT-PCR Assays for the Quantification of Human Enteric Viruses in Environmental Samples.
abstract: Human enteric viruses are responsible for waterborne and shellfish-associated disease outbreaks worldwide. Quantitative reverse transcription PCR (qRT-PCR) is often used to assess the health risks associated with shellfish and environmental water, but viral titres in sediments are less commonly investigated. In this study, we developed and validated two multiplex qRT-PCR assays for aquatic sediment and shellfish samples targeting viruses that are a common cause of gastroenteritis (norovirus GI, GII and hepatitis A virus), two emerging viruses (sapovirus and hepatitis E virus), along with mengovirus (MgV), which is often used as a sample process control for the assessment of RNA extraction efficiency. Singleplex and multiplex assays demonstrated comparable PCR efficiencies and gave reliable results over a wide concentration range. The multiplex assays showed remarkable sensitivity with a limit of detection of 1 RNA copy/µL nucleic acid extract for all target viruses and limits of quantification of 3-18 RNA copies/µL for the targeted human pathogenic viruses and 20-40 RNA copies/µL for MgV. The results demonstrated the veracity of multiplex qRT-PCR for the estimation of viral titres in sediment and shellfish, allowing the rapid assessment of viral infection risks associated with environments exposed to wastewater contamination.
authors.affiliation: School of Environment, Natural Resources and Geography, Bangor University, Deiniol Road, Bangor, Gwynedd, LL57 2UW, UK. fkata211@gmail.com.
authors.forename: Kata
authors.initials: K
authors.lastname: Farkas
authors.affiliation: School of Medical Sciences, Bangor University, Brigantia Building, Penrallt Road, Bangor, Gwynedd, LL57 2AS, UK.
authors.forename: Dafydd E
authors.initials: DE
authors.lastname: Peters
authors.affiliation: School of Biological Sciences, Bangor University, Deiniol Road, Bangor, Gwynedd, LL57 2UW, UK.
authors.forename: James E
authors.initials: JE
authors.lastname: McDonald
authors.affiliation: Centre National de Référence Des Virus Entériques, Laboratoire de Virologie-Sérologie, CHU de Dijon, 2 rue Angélique Ducoudray, BP 37013, 21070, Dijon Cedex, France.|UMR PAM A 02.102 Procédés Alimentaires et Microbiologiques, Université de Bourgogne Franche-Comté/AgroSup Dijon, 1 Esplanade Erasme, 21000, Dijon, France.
authors.forename: Alexis
authors.initials: A
authors.lastname: de Rougemont
authors.affiliation: School of Ocean Sciences, Bangor University, Menai Bridge, Anglesey, LL59 5AB, UK.
authors.forename: Shelagh K
authors.initials: SK
authors.lastname: Malham
authors.affiliation: School of Environment, Natural Resources and Geography, Bangor University, Deiniol Road, Bangor, Gwynedd, LL57 2UW, UK.
authors.forename: Davey L
authors.initials: DL
authors.lastname: Jones
mesh_terms: D004770:Enterovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D004769:Enterovirus Infections / Q000821:virology*; D005506:Food Contamination / Q000032:analysis*; D019015:Geologic Sediments / Q000821:virology*; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012758:Shellfish / Q000821:virology*; D062065:Wastewater / Q000821:virology*
chemical_list: D062065:Wastewater
keywords: Enteric viruses; Multiplex real-time reverse transcription PCR; Nucleic acid quantification; Sediment; Shellfish