Evaluation of a simple and cost effective filter paper-based shipping and storage medium for environmental sampling of F-RNA coliphages

Abstract

Male specific RNA (F-RNA) coliphages are used as indicators of fecal contamination and for source tracking. However, collecting fecal samples for analysis from remote sites is problematic due to the need for an uninterrupted cold chain to guarantee sample suitability for downstream molecular detection of these coliphages. Here, we investigated the feasibility of using filter paper as a collection and storage vehicle for F-RNA coliphages. Various concentrations (10(1) to 10(4)pfu) of two F-RNA coliphages, MS2 and Qβ, were prepared in lambda buffer or a 10% bovine manure slurry, spotted onto filter paper disks, dried, and maintained at 37 °C for up to 37 days. Nucleic acids were extracted from the spotted filter paper disks at 0, 6, 13, and 37 days post inoculation and analyzed by real time RT-PCR. F-RNA coliphages at concentrations of 10(2)pfu/filter paper unit were readily detected, and only a slight decrease in nucleic acid detection was observed over time. Furthermore, the sensitivity of real time RT-PCR detection of the F-RNA coliphage RNA was similar between the developed filter paper sampling methodology and traditional cold storage. These results indicate that filter paper is a suitable storage and transport medium for F-RNA coliphages when refrigeration is not possible.

Keywords: F-RNA coliphages; Fecal indicators; Filter paper; Real time RT-PCR; Source tracking.