Development and Application of Real-Time PCR Assay for Quantification of Mycobacterium ulcerans DNA

Abstract

Buruli ulcer, an infection caused by Mycobacterium ulcerans, is, after tuberculosis and leprosy, the third most common mycobacterial disease. The mode of transmission of M. ulcerans is not exactly known, but since Buruli ulcer often occurs in focalized swampy areas, it is assumed that there is a reservoir of the pathogen in stagnant water. Buruli ulcer usually starts as a painless nodule and can lead to massive destruction of skin, subcutaneous tissue, and eventually muscle and bone. Currently the only recommended treatment is wide surgical excision. In this report we describe the development of a real-time PCR method for the quantification of M. ulcerans DNA (IS2404 TaqMan). The highly specific assay is based on the detection of the M. ulcerans specific insertion sequence IS2404. The IS2404 TaqMan assay turned out to be about 10 times more sensitive than the available conventional PCR-based diagnostic test. It is demonstrated that the IS2404 TaqMan assay is suitable for the quantitative assessment of the dissemination of the mycobacteria in Buruli ulcer lesions. Prototype results obtained with excised tissue from a patient with a late preulcerative Buruli ulcer lesion reconfirmed earlier histopathological findings indicating that tissue damage occurs far beyond the regions in which large numbers of mycobacteria are detectable. The IS2404 TaqMan assay should be a useful tool for both diagnosis and research into the pathology and mode of transmission of this still inadequately investigated mycobacterial disease.

Genetic Diversity inMycobacterium ulceransIsolates from Ghana Revealed by a Newly Identified Locus Containing a Variable Number of Tandem Repeats

“…DNA was extracted as described previously (17). Briefly, small bacterial pellets were heated for 1 h at 95°C in 500 l of an extraction mixture (50 mM Tris-HCl, 25 mM EDTA, 5% monosodium glutamate).…”

Evaluation of Real-Time and Conventional PCR Targeting Complex 85 Genes for Detection of Mycobacterium leprae DNA in Skin Biopsy Samples from Patients Diagnosed with Leprosy

“…Different target sequences for PCR, such as genes encoding the 36-kDa antigen (7), the 18-kDa antigen (20), or the 65-kDa antigen (14) and the repetitive sequences (8) of M. leprae, are available. Recently, real-time PCR technology has improved the diagnosis of many pathogens (9,17). One of the advantages of this method is the quantitation of bacterial DNA content in clinical samples.…”

Rapid Identification of Key Pathogens in Wound Infection by Molecular Means

“…Rapid diagnosis can be achieved by the direct detection of characteristic bacterial genes in clinical specimens. In recent years, real-time PCR has been applied successfully in the medical field, for example in the quantitation of various DNA and RNA viruses in patients [5][6][7][8][9], in the detection of gene amplification [ 10], gene mutations [ 11,12], or chromosomal rearrangements, in the quantitation of gene expression [10,12], and in quantitation of particular pathogens in clinical specimens [13][14][15][16][17][18][19]. Here, we describe the use of real-time PCR (TaqMan) assay for high throughput detection of wound infection pathogens.…”