Multiplex quantitative real-time reverse transcriptase PCR for F+-specific RNA coliphages: a method for use in microbial source tracking

Abstract

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.

FIG. 1.

Negative image of ethidium bromide-stained agarose gel (3%) indicating the specificity of each primer set. I, subgroup I; II, subgroup II; III, subgroup III; IV, subgroup IV. L, pUC 19/HpaII digest double-stranded DNA marker (Ambion).

FIG. 2.

Melting curves demonstrating the specificity of each molecular beacon (▪, matching template; ▿, mismatching template cocktail; ○, no template control). At high temperature the stem hybrid structure of a beacon is dissociated. As the temperature decreases the beacon hybridizes with the specific template (▪), forcing the stem hybrid to remain dissociated and, therefore, producing a fluorescent signal. Treatments with templates of other subgroups (▿) do not exhibit an increase in fluorescence, as the beacon forms closed stem-loop structure.

FIG. 3.

Inactivation of F-specific RNA coliphages in seawater (•, subgroup I infective; ▪, subgroup II; ▾, subgroup III; ▴, subgroup IV) and the quantity of the phage particles detected using the real-time RT-PCR protocol (○, subgroup I; □, subgroup II; ▿, subgroup III; Δ, subgroup IV RT-PCR). Symbols indicate the means, and bars indicate the standard errors. The detection limit of the DAL assay was 1 PFU/ml, which corresponds to −6 on the y axis.

FIG. 4.

Simultaneous quantification of two different contamination sources (▪, raw municipal sewage [subgroup III]; □, chicken stool [subgroup I]) mixed at different ratios. Columns indicate the means, and bars indicate the standard errors.