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.