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

Abstract

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.

Quantitative Distribution of Infectious F-Specific RNA Phage Genotypes in Surface Waters

“…For the further validation of FRNAPHs as potential viral indicators, the differentiation of FRNAPH genotypes is required. Such trials have been performed by applying conventional RT-qPCR assays (7,19,20) or RT-PCR-based typing of isolated plaques using RT-PCR-or reverse line blot (RLB)-based methods (4,5,6,13,17,24,39,40). However, to our knowledge, the quantitative genotyping of infectious FRNAPHs has never been conducted, and thus, the fates of FRNAPH genotypes in the water environment have not been clarified.…”

“…FRNAPHs are further classified into four different genotypes/serotypes, GI to GIV, which are represented by MS2, GA, Q␤, and SP phages, respectively (3). Features such as occurrence in a water environment and stability against water treatment are known to be highly variable among FRNAPH genotypes (4)(5)(6)(7)(8)(9)(10)(11)(12)(13). For instance, GII FRNAPHs tend to be the predominant type in raw municipal wastewater, while GI FRNAPHs tend to be predominant after full-scale municipal wastewater treatment (4,6,7).…”

“…For instance, GII FRNAPHs tend to be the predominant type in raw municipal wastewater, while GI FRNAPHs tend to be predominant after full-scale municipal wastewater treatment (4,6,7). GIV FRNAPHs are rarely found in water samples (5,6,11,13). GI FRNAPHs are highly stable in natural environments (8,10).…”

“…However, RT-qPCR-based assays cannot differentiate between infective and inactive Fϩ coliphages. A method coupling plaque isolation with RT-PCR-or other molecular assay-based genotyping was also applied (4,5,6,13,17,24 To recover FRNAPHs or FRNAPH genotypes present in low numbers, the concentration of FRNAPHs is required. In this regard, a method that can effectively recover FRNAPHs without inactivation is desirable.…”

UV-LEDs Efficiently Inactivate DNA and RNA Coliphages

“…Many enteric viruses have similarities with coliphage viruses having RNA or DNA in their genome [2]. Male-specific RNA coliphages such as MS2 are often used as indicators for monitoring virological quality of water [3].…”