Loop-Mediated Isothermal Amplification for Detection of the 5.8S Ribosomal Ribonucleic Acid Internal Transcribed Spacer 2 Gene Found in Trypanosoma brucei gambiense

Olga V. Nikolskaia Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland.

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Oriel M. M. Thekisoe Unit for Environmental Sciences and Management, North-West University, Potchefstroom, South Africa.

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J. Stephen Dumler Department of Pathology, University of Maryland School of Medicine, Baltimore, Maryland.

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Dennis J. Grab Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland.

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DOI:
https://doi.org/10.4269/ajtmh.15-0288
Volume/Issue:
Volume 96: Issue 2
Page(s):
275–279
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The loop-mediated isothermal amplification (LAMP) assay with its advantages of cost effectiveness, rapidity, and simplicity, has evolved as a sensitive and specific method for the detection of African trypanosomes. Highly sensitive LAMP reactions specific for Trypanosoma brucei rhodesiense or that recognize but do not discriminate between Trypanosoma brucei brucei, T. b. rhodesiense, Trypanosoma brucei gambiense, and Trypanosoma evansi have been developed. A sensitive LAMP assay targeting the T. b. gambiense 5.8S ribosomal RNA internal transcribed spacer 2 (5.8S-ITS2) gene is also available but this assay does not target binding sites that span the CCCA (C3A) (557–560 bps) insertion site that further differentiates T. b. gambiense from T. b. brucei. Here we describe 5.8S-ITS2-targeted LAMP assay that fit these criteria. The LAMP primer sets containing the T. b. gambiense-specific C3A tetranucleotide at the start of the outer forward primer sequences showed high specificity and sensitivity down to at least 0.1 fg T. b. gambiense genomic DNA.

Author Notes

* Address correspondence to Dennis J. Grab, Department of Pathology, The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Ross 656, Baltimore, MD 21205. E-mail: dgrab1@jhmi.edu

Financial support: This research was supported in part by grants from the National Institutes of Health (5R01AI082695 and 1R21AI079282) to Dennis J. Grab.

Authors' addresses: Olga V. Nikolskaia and Dennis J. Grab, Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD, E-mails: onikols1@jhmi.edu and dgrab1@jhmi.edu. Oriel M. M. Thekisoe, Unit for Environmental Sciences and Management, North-West University, Potchefstroom, South Africa, E-mail: oriel.Thekisoe@nwu.ac.za. J. Stephen Dumler, Department of Pathology, University of Maryland School of Medicine, Baltimore, MD, E-mail: sdumler@som.umaryland.edu.

Copyright:
© The American Society of Tropical Medicine and Hygiene 2017
Received:
13 Apr 2015
|
Accepted:
04 Mar 2016
|
Published Online:
08 Feb 2017
Cover The American Journal of Tropical Medicine and Hygiene
The American Journal of Tropical Medicine and Hygiene
Print ISSN:
0002-9637
Online ISSN:
1476-1645
  • 1.

    Blum JA, Neumayr AL, Hatz CF, 2012. Human African trypanosomiasis in endemic populations and travellers. Eur J Clin Microbiol Infect Dis 31: 905–913.

    • Search Google Scholar
    • Export Citation
  • 2.

    Kennedy PG, 2004. Human African trypanosomiasis of the CNS: current issues and challenges. J Clin Invest 113: 496–504.

  • 3.

    Molyneux DH, Pentreath V, Doua F, 1996. African Trypanosomiasis in Man. London, United Kingdom: WB Saunders.

  • 4.

    World Health Organization, 1998. Control and Surveillance of African Trypanosomiasis. Report of a WHO Expert Committee. Technical Report Series No. 881. Geneva, Switzerland: World Health Organization, 114.

    • Search Google Scholar
    • Export Citation
  • 5.

    Bisser S, Lejon V, Preux PM, Bouteille B, Stanghellini A, Jauberteau MO, Buscher P, Dumas M, 2002. Blood-cerebrospinal fluid barrier and intrathecal immunoglobulins compared to field diagnosis of central nervous system involvement in sleeping sickness. J Neurol Sci 193: 127–135.

    • Search Google Scholar
    • Export Citation
  • 6.

    Mori Y, Notomi T, 2009. Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. J Infect Chemother 15: 62–69.

    • Search Google Scholar
    • Export Citation
  • 7.

    Nagamine K, Hase T, Notomi T, 2002. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes 16: 223–229.

    • Search Google Scholar
    • Export Citation
  • 8.

    Surabattula R, Vejandla MP, Mallepaddi PC, Faulstich K, Polavarapu R, 2013. Simple, rapid, inexpensive platform for the diagnosis of malaria by loop mediated isothermal amplification (LAMP). Exp Parasitol 134: 333–340.

    • Search Google Scholar
    • Export Citation
  • 9.

    Mori Y, Nagamine K, Tomita N, Notomi T, 2001. Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun 289: 150–154.

    • Search Google Scholar
    • Export Citation
  • 10.

    Tomita N, Mori Y, Kanda H, Notomi T, 2008. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc 3: 877–882.

    • Search Google Scholar
    • Export Citation
  • 11.

    Qiao YM, Guo YC, Zhang XE, Zhou YF, Zhang ZP, Wei HP, Yang RF, Wang DB, 2007. Loop-mediated isothermal amplification for rapid detection of Bacillus anthracis spores. Biotechnol Lett 29: 1939–1946.

    • Search Google Scholar
    • Export Citation
  • 12.

    Wastling SL, Picozzi K, Kakembo AS, Welburn SC, 2010. LAMP for human African trypanosomiasis: a comparative study of detection formats. PLoS Negl Trop Dis 4: e865.

    • Search Google Scholar
    • Export Citation
  • 13.

    Kuboki N, Inoue N, Sakurai T, Di Cello F, Grab DJ, Suzuki H, Sugimoto C, Igarashi I, 2003. Loop-mediated isothermal amplification for detection of African trypanosomes. J Clin Microbiol 41: 5517–5524.

    • Search Google Scholar
    • Export Citation
  • 14.

    Njiru ZK, Mikosza AS, Matovu E, Enyaru JC, Ouma JO, Kibona SN, Thompson RC, Ndung'u JM, 2008. African trypanosomiasis: sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification (LAMP) of parasite DNA. Int J Parasitol 38: 589–599.

    • Search Google Scholar
    • Export Citation
  • 15.

    Njiru ZK, Mikosza AS, Armstrong T, Enyaru JC, Ndung'u JM, Thompson AR, 2008. Loop-mediated isothermal amplification (LAMP) method for rapid detection of Trypanosoma brucei rhodesiense. PLoS Negl Trop Dis 2: e147.

    • Search Google Scholar
    • Export Citation
  • 16.

    Mansfield JM, Paulnock DM, 2005. Regulation of innate and acquired immunity in African trypanosomiasis. Parasite Immunol 27: 361–371.

  • 17.

    Thekisoe OM, Kuboki N, Nambota A, Fujisaki K, Sugimoto C, Igarashi I, Yasuda J, Inoue N, 2007. Species-specific loop-mediated isothermal amplification (LAMP) for diagnosis of trypanosomosis. Acta Trop 102: 182–189.

    • Search Google Scholar
    • Export Citation
  • 18.

    Agbo EC, Majiwa PA, Claassen EJ, Roos MH, 2001. Measure of molecular diversity within the Trypanosoma brucei subspecies Trypanosoma brucei brucei and Trypanosoma brucei gambiense as revealed by genotypic characterization. Exp Parasitol 99: 123–131.

    • Search Google Scholar
    • Export Citation
  • 19.

    Grab DJ, Nikolskaia OV, Inoue N, Thekisoe MM, Morrison L, Gibson W, Dumler JS, 2011. Using detergent to enhance detection sensitivity of African trypanosomes in human CSF and blood by loop-mediated isothermal amplification (LAMP). PLoS Negl Trop Dis 5: e1249.

    • Search Google Scholar
    • Export Citation
  • 20.

    Aarthy R, Mani S, Velusami S, Sundarsingh S, Rajkumar T, 2015. Role of circulating cell-free DNA in cancers. Mol Diagn Ther 6: 339–350.

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