Development and evaluation of a culture-independent method for source determination of fecal wastes in surface and storm waters using reverse transcriptase-PCR detection of FRNA coliphage genogroup gene sequences

• Published in: Journal of microbiological methods 2015-05, Vol.112, p.28-35
• Main Authors: Paar, Jack, Doolittle, Mark M., Varma, Manju, Siefring, Shawn, Oshima, Kevin, Haugland, Richard A.
• Format: Article
• Language: English
• Subjects: Animals; Bacterial fecal indicators; Coliphages - classification; Coliphages - genetics; Coliphages - isolation & purification; Detection and quantification methods; Feces - virology; FRNA coliphage; Genetic markers; Genotype; Humans; Reverse Transcriptase Polymerase Chain Reaction - methods; Urban stormwater; Water Pollution
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2015.02.015

A method, incorporating recently improved reverse transcriptase-PCR primer/probe assays and including controls for detecting interferences in RNA recovery and analysis, was developed for the direct, culture-independent detection of genetic markers from FRNA coliphage genogroups I, II & IV in water samples. Results were obtained from an initial evaluation of the performance of this method in analyses of waste water, ambient surface water and stormwater drain and outfall samples from predominantly urban locations. The evaluation also included a comparison of the occurrence of the FRNA genetic markers with genetic markers from general and human-related bacterial fecal indicators determined by current or pending EPA-validated qPCR methods. Strong associations were observed between the occurrence of the putatively human related FRNA genogroup II marker and the densities of the bacterial markers in the stormwater drain and outfall samples. However fewer samples were positive for FRNA coliphage compared to either the general bacterial fecal indicator or the human-related bacterial fecal indicator markers particularly for ambient water samples. Together, these methods show promise as complementary tools for the identification of contaminated storm water drainage systems as well as the determination of human and non-human sources of contamination. [Display omitted] •An rtPCR method was developed for detecting FRNA coliphage genogroups in water.•Method performance was tested in analyses of urban surface and storm drain waters.•Method results were compared with general and human fecal bacteria qPCR methods.•FRNA genogroup II and human fecal bacteria co-occurred in many storm drain waters.•FRNA coliphage was infrequently detected in the surface waters.