Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Dec 4;8(12):e3368.
doi: 10.1371/journal.pntd.0003368. eCollection 2014 Dec.

Rapid identification of black grain eumycetoma causative agents using rolling circle amplification

Sarah A Ahmed  1 , Bert H G Gerrits van den Ende  2 , Ahmed H Fahal  3 , Wendy W J van de Sande  4 , G S de Hoog  5
Affiliations

Rapid identification of black grain eumycetoma causative agents using rolling circle amplification

Sarah A Ahmed et al. PLoS Negl Trop Dis. .

Abstract

Accurate identification of mycetoma causative agent is a priority for treatment. However, current identification tools are far from being satisfactory for both reliable diagnosis and epidemiological investigations. A rapid, simple, and highly efficient molecular based method for identification of agents of black grain eumycetoma is introduced, aiming to improve diagnostic in endemic areas. Rolling Circle Amplification (RCA) uses species-specific padlock probes and isothermal DNA amplification. The tests were based on ITS sequences and developed for Falciformispora senegalensis, F. tompkinsii, Madurella fahalii, M. mycetomatis, M. pseudomycetomatis, M. tropicana, Medicopsis romeroi, and Trematosphaeria grisea. With the isothermal RCA assay, 62 isolates were successfully identified with 100% specificity and no cross reactivity or false results. The main advantage of this technique is the low-cost, high specificity, and simplicity. In addition, it is highly reproducible and can be performed within a single day.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Specificity of rolling circle amplification probes.
Agarose gel electrophoresis analysis of rolling circle amplification products. Positives probe signal seen as band pattern was only present with matched template–probe mixtures. Probe names are indicated on the top of the gel. Lanes; 1 M. mycetomatis CBS 109801T, 2 M. tropicana CBS 201.38T, 3 M. pseudomycetomatis CBS 129177T, 4 M. fahalii CBS 129176T, 5 T. grisea CBS 332.50T, 6 F. senegalensis CBS 196.79T, 7 F. tompkinsii CBS 200.70, 8 M. romeroi CBS 252.60T, M DNA ladder.
Figure 2
Figure 2. Madurella mycetomatis identification by RCA.
Gel representation of rolling circle amplification reaction using Madurella mycetomatis probe (MYC) for strains recovered from mycetoma patient of origin: lane 1–18 Sudan; lane 19, 20 Mali; lane 21, 22 India; lane 23 negative control water; lane M ladder.
Figure 3
Figure 3. Identification time of species using rolling circle amplification (RCA) and sequencing of ITS.
See this image and copyright information in PMC

References

    1. Fahal AH (2006) Mycetoma, Clinicopathological Monograph. Khartoum: Khartoum University Press.
    1. van de Sande WW, Maghoub el S, Fahal AH, Goodfellow M, Welsh O, et al. (2014) The mycetoma knowledge gap: identification of research priorities. PLoS Negl Trop Dis. 8: e2667. - PMC - PubMed
    1. Kwon-Chung KJ, Bennet JE (1992) Medical Mycology. Philadelphia: Lea and Febiger.
    1. Fahal AH (2010) Management of mycetoma. Expert Rev Dermatol 5: 87–93.
    1. Yousif BM, Fahal AH, Shakir MY (2010) A new technique for the diagnosis of mycetoma using fixed blocks of aspirated material. Trans R Soc Trop Med Hyg 104: 6–9. - PubMed

MeSH terms