title: Design and assessment of a real time reverse transcription-PCR method to genotype single-stranded RNA male-specific coliphages (Family Leviviridae).
abstract: A real-time, reverse transcription-PCR (RT-qPCR) assay was developed to differentiate the four genogroups of male-specific ssRNA coliphages (FRNA) (family Leviviridae). As FRNA display a trend of source-specificity (human sewage or animal waste) at the genogroup level, this assay provides a tool to help identify the origin of fecal contamination. Primers and probes were designed using complete genomic sequences from 29 FRNA phages. The final selection of primer/probe sets were based on (i) ability to amplify a single, specific product, (ii) genogroup specificity, (iii) lack of cross-reactivity, and (iv) experimental reproducibility and sensitivity over a range of target concentrations. Assay time was reduced by using heat-released viral RNA rather than purified RNA. For quality assurance, a custom RNA molecule was employed as an internal, non-competitive control. The usefulness of this method to identify sources of fecal contamination was tested on a total of 49 FRNA phages isolated from various warm-blooded animals, sewage and combined sewage overflow. FRNA phages from animal wastes were genotyped as 86% I, 4% III Q-like and 9% IV. Two sewage isolates typed to genogroup I and combined sewage overflow isolates genotyped as 40% II and 52% III. Primer specificity designed from this comprehensive sequence database may better discriminate FRNA from different sources.
authors.affiliation: US Environmental Protection Agency, Gulf Ecology Division, Gulf Breeze, FL 32561, USA. friedman.stephanie@epa.gov
authors.forename: Stephanie D
authors.initials: SD
authors.lastname: Friedman
authors.forename: Emilie M
authors.initials: EM
authors.lastname: Cooper
authors.forename: Kevin R
authors.initials: KR
authors.lastname: Calci
authors.forename: Fred J
authors.initials: FJ
authors.lastname: Genthner
mesh_terms: D000818:Animals; D003090:Coliphages / Q000145:classification* / Q000235:genetics*; D005243:Feces / Q000821:virology; D005838:Genotype; D008297:Male; D058889:Molecular Typing; D015345:Oligonucleotide Probes / Q000235:genetics; D012328:RNA Viruses / Q000145:classification* / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012722:Sewage / Q000821:virology; D014773:Virology / Q000379:methods* / Q000592:standards
chemical_list: D015345:Oligonucleotide Probes; D012722:Sewage