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Quantitative CrAssphage PCR Assays for Human Fecal Pollution Measurement
Stachler, E., C. Kelty, Mano Sivaganesan, X. Li, K. Bibby, AND O. Shanks. Quantitative CrAssphage PCR Assays for Human Fecal Pollution Measurement. ENVIRONMENTAL SCIENCE & TECHNOLOGY. American Chemical Society, Washington, DC, 51(16):9146-9154, (2017). https://doi.org/10.1021/acs.est.7b02703
Fecal indicator bacteria can be used to monitor fecal pollution; however, they cannot differentiate human fecal waste from that of other animals. The purpose of this work was to test a whether DNA associated with a newly discovered bacteriophage can be used to track human fecal contamination and differentiate it from other animal waste. Also, a comparison was made between this technique and other established, standardized qPCR assays for human fecal source tracking. This new fecal source tracking technique shows promise in becoming an important water quality management tool. This research is relevant to communities, researchers, and regulators managing land or water contaminated with fecal waste.
Environmental waters are monitored for fecal pollution to protect public health and water resources. Traditionally, general fecal indicator bacteria are used; however, they cannot distinguish human fecal waste from pollution from other animals. Recently, a novel bacteriophage, crAssphage, was discovered by metagenomic data mining and reported to be abundant in and closely associated with human fecal waste. To confirm bioinformatic predictions, 384 primer sets were designed along the length of the crAssphage genome. Based upon initial screening, two novel crAssphage qPCR assays (CPQ_056 and CPQ_064) were designed and evaluated in reference fecal samples and water matrices. The assays exhibited high specificities (98.6%) when tested against a large animal fecal reference library and were highly abundant in raw sewage and sewage impacted water samples. In addition, CPQ_056 and CPQ_064 assay performance was compared to HF183/BacR287 and HumM2 methods in paired experiments. Findings confirm viral crAssphage qPCR assays perform at a similar level to well established bacterial human-associated fecal source identification technologies. These new viral based assays could become important water quality management and research tools.