Comparing DNA Extraction Methods for Environmental Water Samples: A Malawi Case Study

Current standards for fecal contamination in surface waters limit source identification and rapid results from nonpoint, and point sources with human fecal effluent. Microbial source tracking methods have been developed to identify human fecal sources from environmental samples. These methods are highly dependent on the extracted DNA quality and quantity and co-extracted inhibitors which limit downstream applications. In this study, we collected 11 environmental water samples from Malawi, East Africa; 1 tap water sample, 1 waste water sample, 2 lake water samples and 7 river water samples (20 ml – 500 ml). Samples were membrane filtered and stored in the dark until DNA extraction. We compared three extraction protocols on the sample filters; Qiamp Stool DNA Mini Kit® (Qiagen), PowerSoil Kit® (Mo Bio Laboratories) and EPA DNA Crude Extraction Method followed by Qiamp DNA Mini Kit® (Qiagen). The DNA extracts were tested for three markers; Escherichia coli uid gene, Enterococci 23S gene and a human specific marker, Bacteroides thetaiotaomicron alpha mannanese gene, with qPCR.The Enterococci, Bacteriodes and E. coli markers from all samples ranged from 1.7*102 -3.1*104 cells/100ml; 1.5*102 -6.1*104 cells/100ml and 6.8*103 -7*105 cells/100ml, respectively. The kits’ extraction efficacy varied according to the indicator. For all water types, E.coli and Bacteroides markers’ amplification from the three kits was not statistically different (p>0.05). However, the Enterococci marker’s amplification from the EPA Extraction combined with Qiamp was significantly lower than the Qiamp Stool and Mo Bio PowerSoil Kits (p<0/05). For the river samples, the E.coli and Bacteroides markers’ amplifications was significantly higher in Mo Bio PowerWater Kit. The Enterococci marker amplification were significantly higher in the Qiamp Stool Kit (p>0.05). The data indicate that Qiamp Stool kit is better at extracting Enteroocci’s gram + DNA, while Mo Bio PowerSoil is better at extracting Bacteroides. Larger dataset trials will be needed to conclusively decide which protocols work best for various environmental water samples and targets.