Direct Extraction and Assessment of Genomic DNA of Mycetoma Fungi from Black-grains Specimen

  • Elhadi Abdalla Ahmed Faculty of Medical Laboratory Sciences, University of Gezira
  • Bakri Y M Nour BNNICD, University of Gezira, Sudan
  • Adam D Abakar Department of Medical Parasitology, Faculty of Medical Laboratory Sciences, University of Gezira, Sudan
  • Gamal M. A. Ahmed Faculty of Medicine, University of Gezira, Sudan
  • Ahmed E Ahmed Ministry of Health, Gezira State, Sudan

Abstract

Background: Direct isolation of genomic DNA of mycetoma fungi from black-grains achieve rapid diagnosis and may overcome culture disadvantages. Objectives: This study aimed to isolate and assess the DNA of mycetoma fungi using black-grains and to apply amplification of ITS region and nucleotide sequences. Methods: CTAB method was followed by manual homogenization alternatively to liquid nitrogen and glass beads disruption to obtain the genomic DNA. Results: Yielded DNA concentrations vary from 1.50 to 47.97 μg/ml (mean 10.09 μg/ml) while the optimum DNA purity recorded with 75.8% of specimens (n=69/91).Successful amplification of ITS region was done using pan-fungal primers (ITS4/5) with 90.1 (n=82/91)percentage. Species nucleotide sequences were detected with 67 (94.4%) amplicons from a total of 71.Conclusion: The study recommended using of black-grain specimens for DNA extraction of mycetoma fungi parallel with culture to insure rabid diagnosis and identification.

References

1. Desnos-Ollivier M, Bretagne S, Dromer F, Lortholary O, Dannaoui E. Molecular identification of black-grain mycetoma agents. J Clin Microbiol. 2006;44(10):3517-3523. doi:10.1128/JCM.00862-06.
2. van de Sande W, Fahal A, Ahmed SA, Serrano JA, Bonifaz A, Zijlstra E; eumycetoma working group. Closing the mycetoma knowledge gap. Med Mycol. 2018 Apr 1;56(suppl_1):153-164. doi: 10.1093/mmy/myx061. PMID: 28992217.
3. Fredricks DN, Smith C, Meier A. Comparison of six DNA extraction methods for recovery of fungal DNA as assessed by quantitative PCR. J Clin Microbiol. 2005 Oct;43(10):5122-8. doi: 10.1128/JCM.43.10.5122-5128.2005. PMID: 16207973; PMCID: PMC1248488.
4. Umesha, S., Manu kumar, H.M. & Raghava, S. A rapid method for isolation of genomic DNA from food-borne fungal pathogens. Biotech. 2016 Jun;6:(123). DOI 10.1007/s13205-016-0436-4.
5. Ahmed SA, van den Ende BH, Fahal AH, van de Sande WW, de Hoog GS. Rapid identification of black grain eumycetoma causative agents using rolling circle amplification. PLoS Negl Trop Dis. 2014;8(12):e3368. Published 2014 Dec 4. doi:10.1371/journal.pntd.0003368.
6. van de Sande WW, Fahal AH, Good fellow M, Mahgoubel S, Welsh O, Zijlstra EE. Merits and pitfalls of currently used diagnostic tools in mycetoma. PLoS Negl Trop Dis. 2014;8(7):e2918. Published 2014 Jul 3. doi:10.1371/journal.pntd.0002918.
7. van de sende WWJ.et al., Madurella. In Liu D, editor. Molecular detection of human fungal pathogens. Boca Raton; CRC Press, Taylor & Francis Group. 2011: 117-128.
8. Ahmed SA, van de Sande WW, Desnos-Ollivier M, Fahal AH, Mhmoud NA, de Hoog GS. Application of isothermal amplification techniques for identification of Madurella mycetomatis, the prevalent agent of human mycetoma. J Clin Microbiol. 2015;53(10):3280-3285. doi:10.1128/JCM.01544-15.
9. Ahmed A, Adelmann D, Fahal A, Verbrugh H, van Belkum A, de Hoog S. Environmental occurrence of Madurellamycetomatis, the major agent of human eumycetoma in Sudan. J Clin Microbiol. 2002;40(3):1031-1036. doi:10.1128/jcm.40.3.1031-1036.2002.
10. Swapan K. Tripathy et al., Exploringrapid and efficient protocol for isolation of fungal DNA.Int.J.Curr.Microbiol.App.Sci 2017;(63): 951-960.doi: https://doi.org/10.20546/ijcmas.2017.603.113.
11. Watts, CJ, Wagner, DK, Sohnle, P G, in Moselio Schaechter, Editor. Fungal Infections, Cutaneous. Academic Press. P:382-388. https://doi.org/10.1016/B978-012373944-5.00226-1.
12. Cullings KW. Design and testing of a plant-specific PCR primer for ecological and evolutionary studies. Molecular Ecology 1992;1(4):233-240.
13. D Huling et al. Rapid detection of genetically modified ingredients in Soybean products by Real-time loop-mediated isothermal amplification. Journal of Food and Nutrition Research. 2014; 2(7):363-368. doi: 10.12691/jfnr-2-7-6.
14. Doyle J and Dichson EE. Preservation of plant sample for DNA restriction endonuclease analysis. Taxon1987;36(4):7115-722.doi.org/10.2307/1221122.
15. Altschul SF, Madden TL, Schäffer AA, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25(17):3389-3402. doi:10.1093/nar/25.17.3389.
16. Hall TA. “BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT”. Nucl. Acids. Symp. Ser. 1999;(41):95-98.
17. van de Sande WW. Global burden of human mycetoma: a systematic review and meta-analysis. PLoSNegl Trop Dis. 2013; 7: e2550. doi.org/10.1371/journal.pntd.0002550 PMID: 24244780.
18. van Belkum A, Fahal A, van de Sande WW. Mycetoma caused by Madurella mycetomatis: a completely neglected medico-social dilemma. Adv Exp Med Biol. 2013; 764: 179–189. doi.org/10.1007/978-1-4614-4726-9_15 PMID: 23654067.
19. Fahal AH. Mycetoma: Clinical Experience from the Sudan. International mycetoma conference. 2002, Khartoum, Sudan.
20. Branscomb R. Mycetoma: An Overview, Laboratory Medicine, 2003 Nov;34(11):803–808.doi.org/10.1309/9763AYLJQN6KTMPA.
21. Zhang YJ, Zhang S, Liu XZ, Wen HA, Wang M. A simple method of genomic DNA extraction suitable for analysis of bulk fungal strains. Lett Appl Microbiol. 2010 Jul;51(1):114-8. doi: 10.1111/j.1472-765X.2010.02867.x.Epub 2010 May 14. PMID: 20536704.
22. Velegraki A, Kambouris M, Kostourou A, Chalevelakis G, Legakis NJ. Rapid extraction of fungal DNA from clinical samples for PCR amplification. Med Mycol. 1999 Feb;37(1):69-73. PMID: 10200937.
23. Ahmed AO, Desplaces N, Leonard P, Goldstein F, De Hoog S, Verbrugh H, van Belkum A. Molecular detection and identification of agents of eumycetoma: detailed report of two cases. J Clin Microbiol. 2003 Dec;41(12):5813-6. doi: 10.1128/jcm.41.12.5813-5816.2003.
Published
2022-02-03
How to Cite
AHMED, Elhadi Abdalla et al. Direct Extraction and Assessment of Genomic DNA of Mycetoma Fungi from Black-grains Specimen. Gezira Journal of Health Sciences, [S.l.], v. 17, n. 1, p. 24-36, feb. 2022. ISSN 1810-5386. Available at: <http://journals.uofg.edu.sd/index.php/gjhs/article/view/1669>. Date accessed: 04 dec. 2022.
Section
Articles