Use of Direct-qPCR for Quanitification of DNA Markers in Microbial Source Tracking Studies of Three Utah Watersheds

Although many studies have successfully evaluated the level of fecal contamination in various watersheds, Microbial Source Tracking (MST) methods have increasingly been used to determine the source of microbes in the water. The EPA has no set standard by which MST must be performed; however, qPCR methods have been applied to MST in recent years to eliminate the need to culture or create libraries through the use of species specific primers. The other various methods include library dependent or culture dependent processes such as: DNA fingerprinting and rep-PCR, which take weeks to months to perform and analyze. Using these primers, which amplify 16srRNA regions of Bacterioides in various mammals (Human, Cow and Horse), we have successfully performed direct-qPCR of fecal samples on known origins without DNA extraction with 100% sensitivity, although, cross amplification (low specificity) to non-target samples has occurred. We are currently preparing standards to which we may determine CT values of amplified target and non-target samples as well as quantifying the amount of bacteria contributed by each source. This study will provide a cost effective way to accurately determine both the source of fecal contamination in iUTAH’s three watersheds and quantify the amount of bacteria coming from humans, horses and cows within hours of receiving samples