Evaluation of RT-PCR and reverse line blot hybridization for detection and genotyping F+ RNA coliphages from estuarine waters and molluscan shellfish

Aims: To evaluate a PCR-based detection and typing method for faecal indicator viruses (F+ RNA coliphages) in water and shellfish, and apply the method for better understanding of the ecology and microbial source tracking potential of these viruses. Methods and Results: Water and shellfish samples were collected over 3 years at nine estuaries in the East, West and Gulf Coasts of the USA, providing 1033 F+ RNA coliphage isolates. F+ RNA coliphage genotyping rates by reverse transcriptase-PCR - reverse line blot (RLB) hybridization ranged from 94.7% to 100% among estuaries, and were not significantly different in oysters, clams, mussels or water (P = 0.8427). Twenty samples negative by RLB were nucleotide sequenced for confirmation, and to refine RLB probes. More F+ RNA coliphages were genotyped from colder water than warmer waters, while the water salinity did not affect F+ RNA coliphage levels. Conclusions: RT-PCR-RLB was a robust method for detecting and genotyping F+ RNA coliphages from diverse coastal areas, which provided new information on the ecology of F+ RNA coliphages. Significance and Impact of the Study: This performance-validated F+ RNA coliphage method can be used for faecal indicator monitoring and microbial source tracking, to protect recreational bathers and shellfish consumers from exposure to pathogenic virus and their disease risks.