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Design and assessment of molecular assays to genotype male-specific (F+RNA) coliphages (Family Leviviridae)
Citation:
Friedman, S. Design and assessment of molecular assays to genotype male-specific (F+RNA) coliphages (Family Leviviridae). Water Microbiology/International Water Association Meeting, Chapel Hill, NC, May 15 - 19, 2017.
Impact/Purpose:
Office of Water published a document on implementing the use of a viral indicator of fecal pollution, this viral indicator is the FRNA coliphage. This presentation discusses molecular assays to detect this virus. Identifying the fecal sources of contamination (non-human vs human) through the use of male-specific FRNA coliphage assays provides valuable information for risk management and source mitigation.
Description:
Monitoring programs for recreational waters utilize indicator bacteria concentrations as predictors of sewage-exposure related illness risks. However, most illnesses contracted through exposure to recreational waters may be of viral etiology. Identifying the fecal sources (non-human vs human) also provides valuable information for risk management and source mitigation. Male-specific (FRNA) coliphages are proposed as sensitive enteric viral indicators for source-tracking fecal pollution in environmental waters. Classified as family Leviviridae of two genera, Levivirus and Allolevivirus, and four genogroups (I, II, III, IV) the genogroups provide information regarding animal or human fecal sources. In order to design an assay for molecular identification of specific genogroups, a genomic sequence database of sufficient size must be generated from several FRNA coliphages collected from diverse sources or locations. The complete genome of 21 FRNA strains was sequenced and compared with 11 strains available in GenBank, for a total of 32 Leviviridae strains. From conserved regions within each genogroup, genogroup-specific primer sets were designed for the following: i) reverse-transcription polymerase chain reaction (RT-PCR) assay and ii) reverse-transcription real-time PCR assay (RT-qPCR). These assays were then evaluated successfully on a panel of environmental FRNA strains demonstrating their usefulness to assess the sanitary quality of recreational waters.