| Microbial contamination of coastal waters poses a significant risk to human health through recreational exposure and consumption of shellfish. Current protocols for assessing the magnitude of microbial contamination in coastal waters cannot distinguish between human and non-human sources. Therefore, there is a critical need at local, regional and national levels for a method that allows reliable discrimination of human and non-human microbial contamination (microbial source tracking).; F+ specific RNA coliphages (Leviviridae) have been suggested as a indicator organism for microbial source tracking. Because subgroup II and III are predominantly associated with human, and subgroup I and IV are predominantly associated with non-human waste the ratio of four subgroups of these phages can be used to determine the sources of contamination.; Due to low concentrations of these phages in the environment, the enrichment technique (Method 1601, US Environmental Protection Agency) has been previously recommended and used for detection. Based on the one-step growth experiments conducted, the burst sizes and infection efficiencies are significantly different between several subgroups and therefore the method cannot be used for quantitative analyses.; Therefore, a sensitive, culture-independent method is needed in order to make the coliphage approach a useful tool for microbial source tracking. To address this, a multiplex real-time RT-PCR protocol for the simultaneous detection and quantification of all four subgroups of F-specific RNA coliphages was developed. Four novel primer sets and four molecular beacons labeled with FAM, HEX, CY-5 or Texas Red were designed. No interference between subgroups was detected in multiplexed amplification reactions. The primers and beacons were highly specific. The standard curves were linear for a minimum of six orders of magnitude. The assay is sensitive, achieving detection with as few as ten copies of isolated coliphage RNA.; The sensitivity of the technique was also increased by the ability to detect the non-infective portion of the phages. During the survival experiments the real-time PCR technique was able to quantify and detect coliphages in seawater when culture based assay failed. The decay constant of the infective phage fraction was significantly higher compared to the decay constant of the real-time RT-PCR estimates. While the infectivity was lost at different rates at the subgroup level, the decay constant calculated using the real-time RT-PCR estimates was not significantly different at the subgroup level avoiding environmental based bias. (Abstract shortened by UMI.)... |
