A Novel Droplet Digital PCR Human mtDNA Assay for Global Fecal Source Tracking

Abstract

Human mitochondrial DNA (mtDNA) provides a promising target for microbial source tracking because it is unique to humans and universal across human individuals. We developed a droplet digital PCR (ddPCR) assay, hCYTB484, targeting the cytochrome b gene of the human mtDNA and compared the performance of the hCYTB484 assay with a widely used assay targeting human-associated Bacteroides, the HF183/BacR287 assay. We also defined and validated the analytical limit of detection and analytical lower limit of quantification; these analytical limits determine the concentration levels above which samples are declared to be positive and quantifiable for the target, respectively. We found both assays to be highly specific (95%) against cow and pig feces; however, the hCYTB484 was more sensitive when tested against individual human feces from US, Bangladesh, and Mozambique (100% versus a mean of 51% across the 3 countries). To further compare the performance of the two assays, we utilized a receiver operating characteristic curve, showing that the hCYTB484 marker was widely distributed across human feces from the 3 different geographical regions tested and in higher quantities than the HF183/BacR287 marker. The higher performance of the hCYTB484 marker in individual feces is a desirable characteristic in the detection of fecal pollution from sources to which fewer individuals contribute, such as non-sewered types of sanitation that serve most of Earths population and carry the highest risk of exposure to fecal-oral pathogens.\n\nImportanceThe usefulness of an ideal human-specific, culture- and library-independent marker to the microbial source tracking field is reflected by the numerous efforts to develop such markers; however, thus far, microbial-based markers of this type have exhibited variable source-specificity across geographies and variable sensitivity across scales of fecal waste. Most of the worlds population is served by non-sewered forms of sanitation that also carry the highest risk of exposure to fecal-oral pathogens. This reality underscores the need for markers of human fecal contamination that have high sensitivity in fecal pollution sources to which fewer individuals contribute to, such as fecal sludges found in pit latrines. We show that human mtDNA-based methods can be highly source-specific and highly sensitive in smaller scales of fecal pollution, providing a useful addition to the microbial source tracking toolbox by complementing the variable performance of microbial-based markers.