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 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.

Keywords: Australian native possums; Buruli ulcer; DNA extraction; Mycobacterium ulcerans; paramagnetic beads.

Fig 1

Overview of SPRI-bead-based DNA extraction and purification methods developed for (A) possum excreta specimens; (B) biological swabs taken from possums with suspected M. ulcerans infection; (C) pure mycobacterial culture. All steps are performed at room temperature. GITC, guanidinium isothiocyanate; AA, ammonium acetate; SDS, sodium dodecyl sulfate; EtOH, ethanol; IS2404, insertion sequence 2404; qPCR, quantitative polymerase chain reaction; PBS, phosphate-buffered saline; RT, room temperature.

Fig 2

Development and validation of SPRI-bead-based DNA extraction methods for detecting M. ulcerans. (A) Stability profile of a contrived M. ulcerans-positive possum excreta. DNA extractions of 50 mg of contrived positive sample were performed in triplicate (mean value plotted) at each time interval and tested for the presence of M. ulcerans insertion sequence (IS) 2404 using qPCR. Error bars represent the 95% CI (B). Efficiency of SPRI-bead-based DNA extractions of increasing weights of contrived positive possum excreta, as assessed by IS2404 qPCR. DNA extractions performed in biological triplicate are shown. Dotted red line shows the square of the Spearman’s correlation coefficient. (C) Comparison of Qiagen PowerSoil Pro and a SPRI-bead-based method for DNA extraction from possum excreta. Possum excreta samples collected during surveillance studies were halved and processed by the “gold-standard” PowerSoil Pro Kit and SPRI-bead method. Blue circles show 44 samples with DNA extractions that tested IS2404 positive by both methods. Dotted lines indicate the Ct ≤ 40 cut-off for the IS2404 qPCR, above which samples are interpreted as negative. Open red circles indicate samples not detected by one method. The 30 samples that tested negative by both DNA extraction methods are not shown. (D) Comparison of Ct values for the 44 possum excreta DNA extractions that tested IS2404 qPCR positive by both methods. The single pale blue circle represents a SPRI bead-extracted sample that was PCR inhibited on initial testing but tested positive after dilution of the DNA extract. Black horizontal bars represent the mean IS2404 qPCR Ct value for each method. The null hypothesis (no difference between means) was rejected for P < 0.01 (Wilcoxon matched-pairs signed rank test, two-tailed P value). ***P = 0.0002. (E) Comparison of the Qiagen Blood & Tissue Kit and a SPRI-bead-based method for DNA extraction from possum tissue swabs. Swabs collected from possums were each eluted in PBS, and equal aliquots of eluate were DNA extracted using either the Blood & Tissue Kit or SPRI-bead method. Blue circles show samples with DNA extractions that tested IS2404 positive by both methods. The five samples that tested negative by both DNA extraction methods are not shown. (F) Comparison of the 10 possum swab samples that tested positive by both DNA extraction methods. Black horizontal bars represent the mean IS2404 qPCR Ct value for each DNA extraction method. The null hypothesis (no difference between means) was rejected for P < 0.01 (Wilcoxon matched-pairs signed rank test, two-tailed P value). **P = 0.0020.