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Development of Cross-Assembly Phage PCR-Based Methods for Human Fecal Source Identification
Citation:
Stachler, E., C. Kelty, K. Bibby, AND O. Shanks. Development of Cross-Assembly Phage PCR-Based Methods for Human Fecal Source Identification. Presented at Water Microbiology Conference, Chapel Hill, NC, May 17 - 19, 2016.
Impact/Purpose:
To inform the public.
Description:
Technologies that can characterize human fecal pollution in environmental waters offer many advantages over traditional general indicator approaches. However, many human-associated methods cross-react with non-human animal sources and lack suitable sensitivity for fecal source identification applications. The genome of a newly discovered bacteriophage (~97 kbp), the Cross-Assembly phage or “crAssphage”, assembled from a human gut metagenome DNA sequence library is predicted to be both highly abundant and predominately occur in human feces suggesting that this double stranded DNA virus may be an ideal human fecal pollution indicator. We report the development of two human-associated crAssphage endpoint PCR methods (crAss056 and crAss064). A shotgun strategy was employed where 384 candidate primers were designed to cover ~41 kbp of the crAssphage genome deemed favorable for method development based on a series of bioinformatics analyses. Candidate primers were subjected to three rounds of testing to evaluate assay optimization, specificity, limit of detection (LOD95), geographic variability, and performance in environmental water samples. The top two performing candidate primer sets exhibited 100% specificity (n = 70 individual samples from 8 different animal species), >90% sensitivity (n = 10 raw sewage samples from different geographic locations), LOD95 of 0.01 ng/µL of total DNA per reaction, and successfully detected human fecal pollution in impaired environmental water samples. Data suggests that novel crAssphage genetic indicators are highly abundant in raw sewage, closely associated with human fecal pollution, and readily detectable in impaired environmental waters showing great promise for future fecal source identification applications. This presentation will discuss the design and testing methodology used to identify human-associated crAssphage genetic regions and present evidence warranting future water quality monitoring research.