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Applied and Environmental Microbiology, August 2007, p. 4733-4740, Vol. 73, No. 15
0099-2240/07/$08.00+0     doi:10.1128/AEM.02971-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Development and Application of Two Multiplex Real-Time PCR Assays for the Detection of Mycobacterium ulcerans in Clinical and Environmental Samples ^,

Janet A. M. Fyfe,^1,* Caroline J. Lavender,^1, Paul D. R. Johnson,^1,2 Maria Globan,¹ Aina Sievers,¹ Joseph Azuolas,³ and Timothy P. Stinear⁴

Mycobacterium Reference Laboratory, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, Australia,¹ Department of Infectious Diseases, Austin Health, Heidelberg, Victoria, Australia,² Department of Primary Industries, Attwood, Victoria, Australia,³ Department of Microbiology, Monash University, Clayton, Victoria, Australia⁴

Received 21 December 2006/ Accepted 20 May 2007

Mycobacterium ulcerans is a slow-growing environmental bacterium that causes a severe skin disease known as Buruli ulcer. PCR has become a reliable and rapid method for the diagnosis of M. ulcerans infection in humans and has been used for the detection of M. ulcerans in the environment. This paper describes the development of a TaqMan assay targeting IS2404 multiplexed with an internal positive control to monitor inhibition with a detection limit of less than 1 genome equivalent of DNA. The assay improves the turnaround time for diagnosis and replaces conventional gel-based PCR as the routine method for laboratory confirmation of M. ulcerans infection in Victoria, Australia. Following analysis of 415 clinical specimens, the new test demonstrated 100% sensitivity and specificity compared with culture. Another multiplex TaqMan assay targeting IS2606 and the ketoreductase-B domain of the M. ulcerans mycolactone polyketide synthase genes was designed to augment the specificity of the IS2404 PCR for the analysis of a variety of environmental samples. Assaying for these three targets enabled the detection of M. ulcerans DNA in soil, sediment, and mosquito extracts collected from an area of endemicity for Buruli ulcer in Victoria with a high degree of confidence. Final confirmation was obtained by the detection and sequencing of variable-number tandem repeat (VNTR) locus 9, which matched the VNTR locus 9 sequence obtained from the clinical isolates in this region. This suite of new methods is enabling rapid progress in the understanding of the ecology of this important human pathogen.

Published ahead of print on 25 May 2007.

Supplemental material for this article may be found at http://aem.asm.org/.

These authors contributed equally to this work.