Rapid bacterial DNA extraction from stool samples

Abstract

Stool samples are a common specimen used in a diagnostic setting. Due to the nature of stool samples, which have a large number of PCR inhibitors, extracting PCR‐ready DNA can be difficult. We have developed a rapid, simple protocol using Evogen ONE chemistry to extract DNA from stools. In this protocol, either 100ul of liquid stool was pipetted or a sterile swab was immersed in formed stool to gather sample which was washed with phosphate‐buffered saline. The supernatant was removed. 250ul of Evogen ONE was added to the sample, vortexed and then incubated for 5 minutes at 95°C. Then the sample was centrifuged and the lysate was removed. 1ul of the lysate containing the DNA was used in the following PCR reactions. A universal bacterial primer set was used with SYBR Green (Bio‐Rad) to show a wide variety of bacterial DNA present. Stool samples that were initially spiked with Salmonella enterica to show specific target recovery were amplified with the Salmonella ttr primer set and a ttr specific HyBeacons probe. All DNA extracts demonstrated amplification products. DNA concentration was measured on a NanoDrop (ThermoFisher). The crossing threshold (Ct) for the generic bacterial primer set was on average 13 cycles, and the Ct for Salmonella‐spiked stools was 27. DNA concentration varied from 150 to 600ng/ul which is typical of the sample type. We have shown that the above protocol is fast, easy to use, and produces high‐quality DNA.