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

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. Water and shellfish samples were collected over 3 years...

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Bibliographic Details
Published in:	Journal of applied microbiology Vol. 104; no. 4; pp. 1203 - 1212
Main Authors:	Love, D.C, Vinjé, J, Khalil, S.M, Murphy, J, Lovelace, G.L, Sobsey, M.D
Format:	Journal Article
Language:	English
Published:	Oxford, UK Oxford, UK : Blackwell Publishing Ltd 01-04-2008 Blackwell Publishing Ltd Blackwell Science
Subjects:	Animals Biological and medical sciences coliphage Coliphages - genetics Coliphages - isolation & purification faecal indicator Feces - virology Fundamental and applied biological sciences. Psychology Genotype microbial source tracking Microbiology Mollusca - virology Nucleic Acid Hybridization - methods Phylogeny reverse line blot hybridization Reverse Transcriptase Polymerase Chain Reaction - methods RNA Phages - genetics RNA Phages - isolation & purification shellfish United States Water Microbiology United States Water Molecular hybridization Shellfish RNA Applied microbiology faecal indicator Estuaries Genotype Coliphage Source Virus Polymerase chain reaction Analysis method Feces Microorganism reverse line blot hybridization Detection microbial source tracking Phage
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Summary:	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. 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. 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. 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.
Bibliography:	http://dx.doi.org/10.1111/j.1365-2672.2007.03646.x ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1364-5072 1365-2672
DOI:	10.1111/j.1365-2672.2007.03646.x