Comparative evaluation of duplex and singleplex qPCR assays for detection of human-associated HF183 and general <i>Bacteroides</i> markers and implications for duplexing in digital PCR

Microbial source tracking (MST) using Bacteroidales markers provides information on faecal contamination in environmental waters. The marker BacUni targets a range of sources, while HF183 is primarily associated with human faeces. Duplex quantitative PCR (qPCR) assays allow simultaneous detection of both markers, though competition between primer-probe sets can affect accuracy. Digital PCR (dPCR) offers absolute quantification and higher sensitivity, but multiplexing can be challenging when marker concentrations differ. This study compared HF183 and BacUni quantification across singleplex and duplex qPCR formats and duplex dPCR. Synthetic standards revealed variability due to gBlock design and probe orientation, whereas environmental samples showed minimal format-dependent effects. Duplex qPCR provided comparable results to singleplex, with minor underestimation relative to dPCR. These small differences likely reflect the use of dPCR-quantified standards for qPCR calibration, rather than using DNA concentration-based calibration methods. Low-copy HF183 samples highlighted dPCR's superior sensitivity near detection limits. GoTaq qPCR was the most economical option, especially in duplex format, whereas dPCR offered competitive costs for duplexed samples with the advantage of absolute quantification. These findings demonstrate that duplex qPCR assays reliably quantify HF183 and BacUni in environmental waters, with dPCR serving as a robust complementary method for low-abundance or confirmatory analyses.