Development and validation of a field-compatible workflow for molecular diagnostics of microbial water quality

Lena Piglmann , Lena Campostrini , Alexander K.T. Kirschner , Andreas H. Farnleitner, Claudia Kolm, Georg Reischer 

Molecular diagnostics provide advantages in many areas of water quality analysis compared to cultivation-based methods, yet their implementation remains limited mainly due to reliance on specialized instruments, trained personnel and regulatory barriers. To bridge this gap, we developed and evaluated a rapid and field-compatible workflow for the assessment of microbiological water quality. The approach combines simple, ionic liquid-based DNA extraction directly from water filters with detection by loop-mediated isothermal amplification (LAMP). Performance was assessed using three representative bacterial targets (Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis) and the human-associated faecal marker HF183 in environmental water samples to determine the applicability in resource-limited settings. Each step of the workflow was carefully benchmarked against state-of-the-art molecular diagnostics. First, our sample processing method was evaluated using dilution series of target cells, demonstrating strong linearity and reliable detection down to very low cell numbers. In spiked environmental water matrices, the method performed competitively with a commercial extraction kit and phenol-chloroform-based extraction both of which require bead-beating steps. The evaluated sample processing method was then coupled with LAMP for on-site target detection. This integrated workflow was highly competitive with reference workflows across the entire concentration range of the chosen targets and showed similar process limits of detection. Validation with environmental water samples demonstrated robust performance, rapid detection times, and minimal variability across matrices. Overall, our workflow offers simplicity without compromising applicability and reliability compared to state-of-the-art molecular methods. The results indicate strong potential for rapid on-site molecular testing in resource-limited settings.