A comparison of DNA extraction procedures for the detection of Mycobacterium ulcerans, the causative agent of Buruli ulcer, in clinical and environmental specimens Lies Durnez a,b, ⁎, Pieter Stragier b , Karen Roebben b , Anthony Ablordey b,c , Herwig Leirs a,d , Françoise Portaels b a Evolutionary Ecology Group, Department of Biology, University of Antwerp, Antwerp, Belgium b Mycobacteriology Unit, Department of Microbiology, Institute of Tropical Medicine, Antwerp, Belgium c Bacteriology Department, Noguchi Memorial Institute for Medical Research, Accra, Ghana d Danish Pest Infestation Laboratory, University of Aarhus, Faculty of Agricultural Sciences, Department of Integrated Pest Management, Kongens Lyngby, Denmark abstract article info Article history: Received 23 June 2008 Received in revised form 22 September 2008 Accepted 2 October 2008 Available online 17 October 2008 Keywords: Buruli ulcer Clinical specimens DNA extraction Environmental specimens Modified Boom Mycobacterium ulcerans Mycobacterium ulcerans is the causative agent of Buruli ulcer, the third most common mycobacterial disease in humans after tuberculosis and leprosy. Although the disease is associated with aquatic ecosystems, cultivation of the bacillus from the environment is difficult to achieve. Therefore, at the moment, research is based on the detection by PCR of the insertion sequence IS2404 present in M. ulcerans and some closely related mycobacteria. In the present study, we compared four DNA extraction methods for detection of M. ulcerans DNA, namely the one tube cell lysis and DNA extraction procedure (OT), the FastPrep procedure (FP), the modified Boom procedure (MB), and the Maxwell® 16 Procedure (M16). The methods were performed on serial dilutions of M. ulcerans, followed by PCR analysis with different PCR targets in M. ulcerans to determine the detection limit (DL) of each method. The purity of the extracted DNA and the time and effort needed were compared as well. All methods were performed on environmental specimens and the two best methods (MB and M16) were tested on clinical specimens for detection of M. ulcerans DNA. When comparing the DLs of the DNA extraction methods, the MB and M16 had a significantly lower DL than the OT and FP. For the different PCR targets, IS2404 showed a significantly lower DL than mlsA, MIRU1, MIRU5 and VNTR6. The FP and M16 were considerably faster than the MB and OT, while the purity of the DNA extracted with the MB was significantly higher than the DNA extracted with the other methods. The MB performed best on the environmental and clinical specimens. This comparative study shows that the modified Boom procedure, although lengthy, provides a better method of DNA extraction than the other methods tested for detection and identification of M. ulcerans in both clinical and environmental specimens. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU), the third most common mycobacterial disease in humans after tuberculosis and leprosy (Portaels, 1995; Meyers and Portaels, 2003). This disease is mainly endemic in Central and West Africa, where it affects mostly poor communities (Debacker et al., 2004; Portaels, 1995). Epidemiological evidence strongly associates BU with aquatic ecosystems and M. ulcerans is considered an environmental pathogen (Portaels, 1995). Cultivation of the bacillus from the environment is however difficult to achieve: presently only one fully characterized M. ulcerans isolate has been recovered from an aquatic insect (Portaels et al., 2008). Therefore, at the moment, detection is based on demonstrating the presence by PCR of the insertion sequence IS2404 in M. ulcerans and some closely related Mycobacterium species, namely, M. liflandii, M. pseudoshottsi and the mycolactone- producing M. marinum strains (Stinear et al., 1999; Stragier et al., 2007). This insertion sequence element has been identified in water, fish, aquatic insects, detritus, leeches, crustaceans, mollusks and mosquitoes, suggesting the presence of IS2404 positive mycobacteria in these specimens (Eddyani et al., 2004; Kotlowski et al., 2004; Johnson et al., 2007). Confirmation of the IS2404 positive environmental specimens remains difficult because of the low M. ulcerans DNA concentration present in environmental specimens (Portaels et al., 2008). Only few researchers have used confirmation PCR targeting other regions in the Journal of Microbiological Methods 76 (2009) 152–158 ⁎ Corresponding author. Mycobacteriology Unit, Department of Microbiology, Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium. Tel.: +32 3 247 63 36; fax: +32 3 247 63 33. E-mail address: ldurnez@itg.be (L. Durnez). 0167-7012/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2008.10.002 Contents lists available at ScienceDirect Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth