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Design and Assessment of a Real Time Reverse Transcription-PCR Method to Genotype Single-Stranded RNA Male-Specific Coliphages (Family Leviviridae).
Citation:
FRIEDMAN, S. D., E. M. COOPER, K. R. CALCI, AND F. J. GENTHNER. Design and Assessment of a Real Time Reverse Transcription-PCR Method to Genotype Single-Stranded RNA Male-Specific Coliphages (Family Leviviridae). JOURNAL OF VIROLOGICAL METHODS. Elsevier Science Ltd, New York, NY, 173(2):196-202, (2011).
Impact/Purpose:
This manuscript describes a molecular method, real-time, reverse transcription-PCR to detect viral fecal indicators of sewage and wastewater discharges. These viruses, termed FRNA phages, have been proposed as a fecal indicator of sewage and aid in discriminating whether or not the aquatic system has been impacted by human or agricultural wastes.
Description:
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, 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 source-track 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 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.