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 significant expense 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 purified genomic 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 efforts to halt the spread of BU in Victoria, Australia, with potential for applicability in other endemic countries. Importance: Buruli ulcer (BU) is a neglected tropical skin disease, with an incidence that has dramatically increased in temperate southeastern Australia over the last decade. In southeastern Australia, BU is a zoonosis with native possums the major wildlife reservoir of the causative pathogen, Mycobacterium ulcerans. Infected possums shed M. ulcerans in their excreta, and excreta surveys using PCR to screen for the presence of pathogen DNA are a powerful means to predict future areas of Buruli ulcer risk for humans. However, excreta surveys across large geographic areas require testing of many thousands of samples. The cost of commercial DNA extraction reagents used for preparing samples for PCR testing can thus become prohibitive to effective surveillance. Here, we describe a simple, low-cost method for extracting DNA from possum excreta using paramagnetic beads. The method is versatile and adaptable to a variety of other sample types including swabs collected from possum tissues and pure cultures of mycobacteria.