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A Reverse Transcription-PCR Assay to Distinguish the Four Genogroups of Male-Specific (F+) RNA Coliphages
Citation:
FRIEDMAN, S. D., E. M. Cooper, L. CASANOVA, M. D. SOBSEY, AND F. J. GENTHNER. A Reverse Transcription-PCR Assay to Distinguish the Four Genogroups of Male-Specific (F+) RNA Coliphages. JOURNAL OF VIROLOGICAL METHODS. Elsevier Science Ltd, New York, NY, 159(1):47-52, (2009).
Impact/Purpose:
Development of genogroup-specific primers and a RT-PCR method for genotype identification of FRNA phages.
Description:
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 have source specificity at the human vs animal level. Reverse-transcription PCR (RT-PCR) can be effectively used for source identification if specific primer sets are designed to be capable of identifying all members within each genogroup. Employing a heat-release procedure that eliminated the need for RNA purification, we developed genogroup-specific primers and an RT-PCR method for genotype identification of FRNA phages. Each genogroup-specific primer set was designed from a minimum of 5 to a maximum of 10 strains of complete genome sequences per group using a total genome data base of 30 strains. The four genogroup-specific primer sets generated discrete PCR amplicon sizes from a variety of environmental phage strains. Cross-reactivity with strains from other genogroups was not observed.