Variability analysis of a low-cost paper dipstick nucleic acid extraction method for wastewater surveillance using gage repeatability and reproducibility

Microbial concentration remains a key challenge in developing low-cost, point-of-use wastewater monitoring assays for pandemic preparedness, primarily due to sample heterogeneity and low pathogen load. Paper dipstick-based methods are promising for simplified and low-cost RNA capture and isolation from sub-milliliter sample volumes. However, given the manual nature of the method, variability is a major concern. To evaluate variability introduced by the method type, operator handling, and wastewater samples, we performed a multi-operator two-way analysis of variance (ANOVA) and 'evaluating the measurement process' (EMP) gage repeatability & reproducibility (gage R&R) study. Two-way ANOVA was used to determine whether differences in Ct values across samples, operators, and their interaction were statistically significant. To further assess the source of variation and quantify the repeatability and reproducibility of the assay, a comprehensive EMP gage R&R analysis was conducted. We detected pepper mild mottle virus (PMMoV) and bacteriophage Phi6 in wastewater samples collected from a sewage pumping station at IIT Bombay campus. PMMoV is abundant in human feces, and a reliable control for normalizing pathogen loads in wastewater by population size, while Phi6 spiked in the wastewater samples is a common surrogate for enveloped viruses such as SARS-CoV-2. Our study demonstrates that dipstick method detected changes in PMMoV load associated with change in the population density due to the campus summer break with the total variation lower than the acceptable gage R&R limit of 30%.