The feasibility of improved live-dead distinction in qPCR-based microbial source tracking

- The study addresses the influence of amplicon length and DMSO on viability MST results.
- Increasing amplicon length typically results in lower proportions of intact bacteria in PMA-qPCR.
- The presence of DMSO during exposure to viability dye helps to suppress signals from damaged bacteria.
- Increased efficiency to suppress dead cell signals might help to improve risk assessment.

PCR-based microbial source tracking (MST) has become a useful tool to identify dominant sources of fecal pollution in water. The method has previously been successfully combined with viability PCR (using propidium monoazide) allowing the preferential detection of membrane-intact bacteria. This study aimed at further improving the selectivity for intact cells when targeting host-specific markers in Bacteroidales bacteria. One approach was to increase amplicon sizes that had been shown to be useful for other applications of viability PCR. For this purpose, two different amplicon sizes were compared when targeting either the genus of Bacteroidales or subgroups thereof specifically associated with human and ruminant fecal material. When applied to different environmental samples, the proposed proportion of intact cells could drop by up to 38% (for sewage treatment effluent from 64 to 26%) when targeting longer sequences. Furthermore co-incubation of the viability dye with dimethylsulfoxide (DMSO) was found to be beneficial, although this observation is currently still empirical. When examining signal decay of artificially contaminated unfiltered river water over six weeks, the PMA treatment effect was observed from the beginning, but the ratio of intact and damaged cells remained constant over time with signals disappearing at the same rate independent of PMA treatment. In this instance the contribution of other factors to overall signal decay seemed more important than loss of membrane integrity.