Q-264. Development and Validation of a Sensitive Taqman Real Time PCR Assay for the Quantification of Two Human Polyomaviruses (JCV and BKV) in Human-Associated Waste Samples

S. McQuaig1, T. Scott2, J. Lukasik2, V. Harwood1;
1Univ. of South Florida, Tampa, FL, 2Biological Consulting Services of North Florida, Gainesville, FL.

Since 1972, each State has been required to set total maximum daily loads (TMDL) for water bodies that do not meet water quality standards according to section 303(d) of the Clean Water Act. A plan of action (TMDL implementation) is then developed and employed to reduce target contaminants. In order to reduce contaminant loading the source(s) must be identified. Microbial source tracking (MST) methods can be employed to determine sources of fecal contamination in water, including assessment of the presence of human polyomaviruses (HPyVs) as an indicator of human-associated fecal pollution. HPyVs are host-specific, excreted in urine, and ubiquitous throughout the population. We have developed and optimized a Taqman quantitative PCR (QPCR) to quantify and simultaneously detect two HPyVs, JCV and BKV. Primers and a probe specific for the conserved T-antigen of both JCV and BKV were used in a QPCR reaction to detect and quantify HPyVs. The QPCR developed was able to consistently detect >10 viruses. To test for specificity, DNA extracted from over 50 animal fecal samples (dog (n=33), cat (n=5), cow (n=6), horse (n=8), sparrow (n=3), duck (n=1)) was analyzed for the presence of HPyVs. HPyVs were not detected in any animal waste samples. To assess the prevalence of HPyVs in human waste, samples were collected from wastewater treatment plants (n=32) and analyzed for HPyVs. DNA was extracted directly from the waste sample. HPyVs were detected in 100% of the human waste samples analyzed. The concentration of HPyVs ranged from 0.7-5.8 x104 viruses per ml of sample. In addition, Escherichia coli, enterococci, and fecal coliforms were enumerated by membrane filtration and standard culture methods. The concentration of bacteria ranged from to 0.1-2.0 x105 CFU/ml. This is the first study to use a QPCR to simultaneously detect both JCV and BKV. The use of this assay in water quality research will allow for a rapid, quantitative, cost effective, and sensitive assessment of water quality and potentially protect humans from exposure to pathogens as well as help officials to identify sources of water pollution for improved remediation of contaminated environmental waters.