Development and application of two multiplex real-time PCR assays for the detection of Mycobacterium ulcerans in clinical and environmental samples

Abstract

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.

FIG. 1.

Standard curve generated using a logarithmic scale by the analysis of known amounts of genomic M. ulcerans DNA with the IS2404, IS2606, and KR TaqMan real-time PCR assays. Each 10-fold dilution was performed in quadruplicate, and the means of these replicates were used as data points. One standard deviation on either side of the mean is shown. The regression lines calculated for the data points for the IS2404, IS2606, and KR assays were as follows: y = −1.1892Ln(x) + 29.156, y = −1.3043Ln(x) + 32.886, and y = −1.428Ln(x) + 32.174, respectively. For each assay, the coefficient of correlation was greater than 0.99.

FIG. 2.

Standard curve generated using a logarithmic scale by the analysis of DNA extracted from pools of 15 Aedes camptorhynchus mosquitoes spiked with known numbers of M. ulcerans organisms with the IS2404, IS2606, and KR TaqMan real-time PCR assays. Each 10-fold dilution was performed in quadruplicate, and the means of these replicates were used as data points. One standard deviation on either side of the mean is shown.