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

Abstract

In Australia, native possums are an animal reservoir for Mycobacterium ulcerans, the causative agent of Buruli ulcer, a neglected tropical skin disease that can progress to extensive ulceration with deformity and disability. Surveillance of possum excreta for shedding of M. ulcerans can be used to inform geospatial modelling to predict locations at which humans are at increased risk of disease acquisition. This discovery provides opportunities to disrupt transmission pathways. However, the significant expense of commercial kits used for DNA extraction from environmental samples in large scale surveillance studies can hinder implementation of public health measures. To address this barrier, we developed a low-cost method for extraction of nucleic acids from possum excreta, possum tissue swabs, and mycobacterial cultures, using a guanidinium thiocyanate lysis solution and paramagnetic beads for DNA clean-up. In 96-well plate format for high-throughput processing, the SPRI-bead DNA extraction method for possum excreta was 3-fold less sensitive but only 1/6 the cost of a widely used commercial kit. While a SPRI-bead microtube-based extraction method for tissue swabs sampled from possums was 4-fold more sensitive and 1/5 the cost of the corresponding commercial kit. Furthermore, when used for preparing DNA from pure mycobacterial cultures, the SPRI-bead method produced genomic DNA with quality metrics comparable to more laborious techniques. The methods described here provide an economical means to continue large-scale M. ulcerans environmental surveillance that should facilitate efforts to halt the spread of Buruli ulcer in Victoria, Australia, with potential for applicability in other endemic countries. IMPORTANCEBuruli ulcer is a neglected tropical skin disease, with an incidence that has dramatically increased in temperate southeastern Australia over the last decade. In this region of the world Buruli ulcer is a zoonosis, Australian native possums are a major wildlife reservoir of the causative agent, Mycobacterium ulcerans, and mosquitoes the vector to humans. 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 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.