Open Access Highly Accessed Research article

Development of Loop-Mediated Isothermal Amplification (LAMP) Assays for Rapid Detection of Ehrlichia ruminantium

Ryo Nakao1, Ellen Y Stromdahl2, Joseph W Magona13, Bonto Faburay114, Boniface Namangala5, Imna Malele6, Noboru Inoue7, Dirk Geysen8, Kiichi Kajino1, Frans Jongejan109 and Chihiro Sugimoto1*

Author Affiliations

1 Department of Collaboration and Education, Research Center for Zoonosis Control, Hokkaido University, Kita 20, Nishi 10, Kita-ku, Sapporo, Hokkaido 001-0020, Japan

2 Entomological Sciences Program, U.S. Army Public Health Command (Provisional), Aberdeen Proving Ground, Maryland 21010-5403, USA

3 National Livestock Resources Research Institute (NaLIRRI), P.O. Box 96, Tororo, Uganda

4 International Trypanotolerance Centre, PMB 14, Banjul, The Gambia

5 Department of Paraclinical Studies, School of Veterinary Medicine, University of Zambia, P.O. Box 32379, Lusaka, Zambia

6 Tsetse & Trypanosomiasis Research Institute, P.O. Box 1026, Tanga, Tanzania

7 National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-13, Inada-cho, Obihiro, Hokkaido 080-8555, Japan

8 Prince Leopold Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium

9 Utrecht Centre for Tick-borne Diseases (UCTD), Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584CL, Utrecht, The Netherlands

10 Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, 0110, Onderstepoort, South Africa

11 Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA

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BMC Microbiology 2010, 10:296  doi:10.1186/1471-2180-10-296

Published: 19 November 2010

Abstract

Background

The rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater, a potential zoonotic disease of ruminants transmitted by ticks of the genus Amblyomma. The disease is distributed in nearly all of sub-Saharan Africa and some islands of the Caribbean, from where it threatens the American mainland. This report describes the development of two different loop-mediated isothermal amplification (LAMP) assays for sensitive and specific detection of E. ruminantium.

Results

Two sets of LAMP primers were designed from the pCS20 and sodB genes. The detection limits for each assay were 10 copies for pCS20 and 5 copies for sodB, which is at least 10 times higher than that of the conventional pCS20 PCR assay. DNA amplification was completed within 60 min. The assays detected 16 different isolates of E. ruminantium from geographically distinct countries as well as two attenuated vaccine isolates. No cross-reaction was observed with genetically related Rickettsiales, including zoonotic Ehrlichia species from the USA. LAMP detected more positive samples than conventional PCR but less than real-time PCR, when tested with field samples collected in sub-Saharan countries.

Conclusions

Due to its simplicity and specificity, LAMP has the potential for use in resource-poor settings and also for active screening of E. ruminantium in both heartwater-endemic areas and regions that are at risk of contracting the disease.