title: Effective detection of human noroviruses in Hawaiian waters using enhanced RT-PCR methods.
abstract: The current recreational water quality criteria using growth-based measurements of fecal indicator bacteria (FIB) concentration have their limitations for swimmer protection. To evaluate the possible use of enteric viruses as an improved indicator of human sewage contamination in recreational waters for enhanced health risk assessment, human norovirus (huNoV) was tested as a model in this study. To establish a highly sensitive protocol for effective huNoV detection in waters, 16 published and newly designed reverse transcription polymerase chain reaction (RT-PCR) primer pairs specific for huNoV genogroup I (GI) and genogroup II (GII) were comparatively evaluated side-by-side using single sources of huNoV RNA stock extracted from local clinical isolates. Under optimized conditions, these RT-PCR protocols shared a very different pattern of detection sensitivity for huNoV. The primer sets COG2F/COG2R and QNIF4/NV1LCR were determined to be the most sensitive ones for huNoV GII and GI, respectively, with up to 10(5)- and 10(6)-fold more sensitive as compared to other sets tested. These two sensitive protocols were validated by positive detection of huNoV in untreated and treated urban wastewater samples. In addition, these RT-PCR protocols enabled detection of the prevalence of huNoV in 5 (GI) and 10 (GII) of 16 recreational water samples collected around the island of O'ahu, which was confirmed by DNA sequencing and sequence analysis. Findings from this study support the possible use of enteric viral pathogens for environmental monitoring and argue the importance and essentiality for such monitoring activity to ensure a safe use of recreational waters.
authors.affiliation: Department of Public Health and Sciences and Microbiology, University of Hawaii at Manoa, Honolulu, Hawaii 96822, USA.
authors.forename: Hsin-I
authors.initials: HI
authors.lastname: Tong
authors.forename: Christina
authors.initials: C
authors.lastname: Connell
authors.forename: Alexandria B
authors.initials: AB
authors.lastname: Boehm
authors.forename: Yuanan
authors.initials: Y
authors.lastname: Lu
mesh_terms: D001483:Base Sequence; D001493:Bathing Beaches; D002947:Cities; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D004777:Environment; D004784:Environmental Monitoring / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification; D005843:Geography; D006254:Hawaii; D006801:Humans; D057230:Limit of Detection; D008969:Molecular Sequence Data; D029322:Norovirus / Q000235:genetics* / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D014865:Waste Disposal, Fluid; D014871:Water Microbiology
chemical_list: D004269:DNA, Bacterial