Evaluation of a Method for Preservation of Surface Water Samples for Analysis of Microbial DNA
thesisposted on 01.08.2021, 00:00 authored by Caroline C Hammond
As of 2021 nearly 30% of the world’s population has inadequate access to clean water. Many resource-poor communities rely on surface water sources like rivers and lakes for daily use, possibly exposing these populations to waterborne illnesses. The wide variety of waterborne pathogens makes it difficult to measure them directly, so indicator species are used to monitor water quality instead. Molecular analyses of water samples can provide useful information about microbes and sources. However, the preservation and transport of environmental water requires freezing at -40C, which makes it difficult to conduct important molecular analyses of water samples from rural areas of low-and middle-income countries. Whatman FTA cards are compact and easy to transport, able to preserve nucleic acid at ambient temperature. Use of Whatman cards to collect environmental water samples would allow greater access to rural communities and molecular analyses for faster and more precise detection of pathogens. However, Whatman cards were originally designed for use with clinical samples, and have yet to be evaluated for their ability to preserve microbial DNA from environmental water samples. This research is an evaluation of Whatman cards for this purpose through three trials, comparing the performance of Whatman FTA cards versus polycarbonate filters. First, recovery and range finding for detection of microbial DNA was done. Next, preservation of known concentrations of E. faecalis cells for varying lengths of time was evaluated. Finally, preservation of wastewater effluent samples for varying lengths of time on Whatman FTA cards and polycarbonate filters was evaluated. All samples were evaluated via quantitative PCR and statistical analyses conducted using SAS version 9.4. After our data was determined to have a non-normal distribution, median and range were used to best describe these data. Each storage media type (polycarbonate, Whatman card)at each specific sample concentration confirmed that each sample type was stable overtime, with polycarbonate samples showing a slight decrease in concentrations as storage length increased. Using the Kruskal-Wallis nonparametric comparison test for group comparisons showed that Whatman cards had similar stability across sample concentrations and storage lengths to polycarbonate filter samples stored at -80oC. Based on this research, Whatman cards present an alternative method of microbial DNA preservation from surface water samples, with less limitations in sample storage and transport than current methods. Samples can be stored for at least two months at ambient temperatures without experiencing substantial nucleic acid degradation, though compared to polycarbonate filters at room temperature, Whatman cards produce more consistent Ct values at concentrations of 1 x 104 cells/mL and above.