Quantitative CrAssphage PCR Assays for Human Fecal Pollution Measurement

Abstract

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 other animal pollution sources. 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 on 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 an animal fecal reference library, and crAssphage genetic markers were highly abundant in raw sewage and sewage-impacted water samples. In addition, CPQ_056 and CPQ_064 performance was compared to HF183/BacR287 and HumM2 assays in paired experiments. Findings confirm that viral crAssphage qPCR assays perform at a similar level to well-established bacterial human-associated fecal-source-identification approaches. These new viral-based assays could become important water quality management and research tools.

Figure 1.

Map representation of the crAssphage genome. The outermost track represents the open reading frames (ORFs) on the forward and reverse strand of the crAssphage genome. The middle track represents the areas of the crAssphage genome that were eliminated from primer design, including noncoding regions, metaviromic islands, modular junction areas, nontarget sequence homology, and regions unsuitable for primer design. The innermost track represents the location of the 384 end-point primer pairs designed in this study and their amplification products.

Figure 2.

Abundance of crAssphage and bacterial human-associated qPCR targets in primary influent sewage (panel A) and environmental water samples (panel B). Values are reported as mean log₁₀ copies estimates per nanogram of total DNA (panel A) or per reaction (panel B) with 95% credible intervals. Sewage qPCR reactions contained 1 ng of template DNA extracted from 10 mL of wastewater, while environmental water qPCR reactions contained 2 μL of DNA extracted from a total volume of 200 mL of impacted water.