A low-cost and versatile paramagnetic bead DNA extraction method for Mycobacterium ulcerans environmental surveillance

Abstract

In Australia, native possums are a major wildlife reservoir for Mycobacterium ulcerans, the causative agent of the neglected tropical skin disease Buruli ulcer (BU). Large-scale possum excreta surveys that use PCR to detect M. ulcerans in 100-1,000 s of excreta specimens are an important tool that can inform geospatial modeling and predict locations of future human BU risk. However, the significantexpense of commercial kits used to extract DNA from specimens is a major barrier to routine implementation. Here, we developed a low-cost method for DNA extraction from possum excreta, possum tissue, and pure mycobacterial cultures, using a guanidinium isothiocyanate lysis solution and paramagnetic beads. In a 96-well plate format for high-throughput processing, the paramagnetic bead DNA extraction method was threefold less sensitive but only 1/6 the cost of a commonly used commercial kit. Applied to tissue swabs, the method was fourfold more sensitive and 1/5 the cost of a commercial kit. When used for preparing DNA from pure mycobacterial cultures, the method yielded purifiedgenomic DNA with quality metrics comparable to more lengthy techniques. Our paramagnetic bead method is an economical means to undertake large-scale M. ulcerans environmental surveillance that will directly inform effortsto halt the spread of BU in Victoria, Australia, with potential for applicability in other endemic countries.