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Applied and Environmental Microbiology, February 2007, p. 808-814, Vol. 73, No. 3
0099-2240/07/$08.00+0     doi:10.1128/AEM.00399-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Multiplex Quantitative Real-Time Reverse Transcriptase PCR for F⁺-Specific RNA Coliphages: a Method for Use in Microbial Source Tracking ^,

Biological Sciences, University of Rhode Island, Kingston,¹ Graduate School of Oceanography, University of Rhode Island, Narragansett, Rhode Island²

Received 17 February 2006/ Accepted 20 November 2006

It is well documented that microbial contamination of coastal waters poses a significant risk to human health through recreational exposure and consumption of shellfish. Identifying the source of microbial contamination (microbial source tracking) plays a dominant role in enabling effective management and remediation strategies. One method used to determine the source of the contamination is quantification of the ratio of the four subgroups of F⁺-specific RNA coliphages (family Leviviridae) in impacted water samples. Because of typically low concentrations in the environment, enrichment assays are performed prior to detection, even though differential replication rates have been reported. These assays are also compromised by differential loss of phage infectivity among subgroups after release into the environment, thus obscuring the initial ratio. Here, a culture-independent multiplex real-time reverse transcriptase-PCR (RT-PCR) protocol for the simultaneous quantification of all four subgroups of F⁺-specific RNA coliphages using novel primer sets and molecular beacons is presented. This assay is extremely sensitive, achieving detection with as few as 10 copies of isolated coliphage RNA, and is linear for a minimum of six orders of magnitude. During survival experiments, the real-time RT-PCR technique was able to quantify coliphages in seawater when culture-based double agar layer assay failed. While infectivity was lost at different rates at the subgroup level, decay constants in seawater, calculated using the real-time RT-PCR estimates, did not vary among subgroups. The accurate determination of the in situ concentration of F⁺-specific RNA coliphages using this method will facilitate more effective remediation strategies for impacted environments.

Published ahead of print on 1 December 2006.

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