Abstract
Human mitochondrial DNA provides a promising target for fecal source tracking because it is unique and intrinsic to humans. We developed a TaqMan chemistry assay, hCYTB484, targeting the cytochrome b gene of the human mitochondrial genome on a droplet digital PCR (ddPCR) platform and compared the performance of hCYTB484 with the HF183/BacR287 assay, a widely used assay targeting human-associated Bacteroides. For both assays, we defined the analytical limit of detection and analytical lower limit of quantification using frequency of detection and imprecision goals, respectively. We then established these analytical limits using empirical ddPCR data, presenting a novel approach to determining the analytical lower limit of quantification. We evaluated assay sensitivity using individual human feces from US, Bangladesh, and Mozambique and evaluated assay specificity using cow, pig, chicken, and goat samples collected from the US. To compare assay performance across a range of thresholds, we utilized receiver operating characteristic curves. The hCYTB484 marker was detected and quantifiable in 100% of the human feces from the 3 geographical distant regions whereas the HF183/BacR287 marker was detectable and quantifiable in 51% and 31% (respectively) of human feces samples. The hCYTB484 marker also was more specific (97%), having fewer detections in pig, chicken, and goat samples than the HF183/BacR287 marker (80%). The higher performance of the hCYTB484 marker in individual feces from geographically distant regions is desirable in the detection of fecal pollution from sources to which fewer individuals contribute, such as the non-sewered forms of sanitation (e.g. pit latrines and septic tanks) that serve most of Earth’s population and carry the highest risk of exposure to fecal-oral pathogens.
Highlights
Word Count 3854/6000 Introduction Within the microbial source tracking (MST) field, the development of an ideal human-specific, culture- and library-independent marker has been a goal for many research efforts for a variety of reasons—notably, its potential as a relatively rapid and broadly-applicable method not requiring the extent of local validation of the host and marker relationship that is required in library43 dependent methods (1–4)
The droplet digital PCR (ddPCR) concentration in a well represents the number of 130 copies of the target found in the 20 microliter PCR reaction; we will refer to copies of target per 131 ddPCR well, which is the ddPCR concentration in a well described above multiplied by 20 μL
We interpolated the level of concentration that would allow us to achieve a coefficient of variation (CV) of 25% by fitting a linear regression to log10 transformed CV and copies per ddPCR well values
Summary
Word Count 3854/6000 (should not exceed approximately 6,000 words, exclusive of methods, references, figure legends, tables, and supplemental material) Introduction Within the microbial source tracking (MST) field, the development of an ideal human-specific, culture- and library-independent marker has been a goal for many research efforts for a variety of reasons—notably, its potential as a relatively rapid and broadly-applicable method not requiring the extent of local validation of the host and marker relationship that is required in library dependent methods (1–4). Microbial DNA markers have exhibited variable performance across different scales of fecal pollution: high sensitivity in wastewater collected from sewage networks but lower sensitivity in human fecal samples (10). Disproportionalities in sanitation exist: the same geographic regions that contain the lowest rates of sewered households are where most of the human population live and where the most human feces are produced (12) This means that for much of the world’s population, enteric pathogen exposure risks are related to sludges and other non-wastewater fecal waste streams that are composites of feces from relatively few individuals. One assay was implemented as a mismatch amplification mutation assay by intentionally designing penultimate primer mismatches to increase species-specificity (13), potentially inducing PCR inefficiencies and leading to increased noise on ddPCR (19) Another assay (14) contained a probe that spanned several population-variable polymorphisms (20–24). We hypothesized that by targeting a widely conserved host-mtDNA marker, we would develop an assay that is more sensitive in human feces from various geographies than the microbial-based HF183/BacR287 assay
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