title: A reverse transcription-PCR assay to distinguish the four genogroups of male-specific (F+) RNA coliphages.
abstract: Goals of reducing fecal contamination in recreational, drinking, shellfishing and other waters and accurately assessing risk from exposure can best be attained if tools to distinguish between sources of pollution are available. The male-specific RNA coliphage (FRNA) genogroups display a trend of source specificity. Reverse transcription-PCR (RT-PCR) can be effectively used for genotyping if specific primer sets are designed to be capable of identifying all members within each genogroup. In this study genogroup-specific primer sets were designed using a minimum of 5 to a maximum of 10 complete phage genome sequences from strains in each genogroup. With these primers and employing a heat-release procedure that eliminated the need for RNA purification an RT-PCR method for genotype identification of FRNA phages was developed. The four genogroup-specific primer sets generated discrete PCR amplicon sizes from a variety of environmental FRNA phage strains. Limits of detection, cross-reactivity and/or non-specific binding to strains from other genogroups were evaluated.
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: Lisa
authors.initials: L
authors.lastname: Casanova
authors.forename: Mark D
authors.initials: MD
authors.lastname: Sobsey
authors.forename: Fred J
authors.initials: FJ
authors.lastname: Genthner
mesh_terms: D001483:Base Sequence; D003090:Coliphages / Q000235:genetics / Q000302:isolation & purification*; D017124:Conserved Sequence; D017931:DNA Primers; D004784:Environmental Monitoring / Q000379:methods; D014644:Genetic Variation; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013045:Species Specificity
chemical_list: D017931:DNA Primers; D012367:RNA, Viral