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Journal of Clinical Microbiology, March 2009, p. 645-651, Vol. 47, No. 3
0095-1137/09/$08.00+0     doi:10.1128/JCM.01412-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Development and Evaluation of a Real-Time Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Rift Valley Fever Virus in Clinical Specimens{triangledown}

C. A. Le Roux,1,3 T. Kubo,2,4 A. A. Grobbelaar,1 P. Jansen van Vuren,1 J. Weyer,1 L. H. Nel,3 R. Swanepoel,1 K. Morita,4 and J. T. Paweska1,5*

Special Pathogens Unit, National Institute for Communicable Diseases of the National Health Laboratory Services, Private Bag X4, Sandringham 2131, Republic of South Africa,1 Center for International Collaborative Research, Institute for Tropical Medicine, Nagasaki University, 1-12-4, Sakamoto, Nagasaki 852-8523, Japan,2 Department of Microbiology and Plant Pathology, Faculty of Natural and Agriculture Sciences, University of Pretoria, Pretoria 0002, Republic of South Africa,3 Department of Virology, Institute of Tropical Medicine, Nagasaki University, 1-12-4, Sakamoto, Nagasaki 852-8523, Japan,4 Division of Virology and Communicable Disease Surveillence, School of Pathology, University of the Witwatersrand, Johannesburg, South Africa5

Received 23 July 2008/ Returned for modification 28 August 2008/ Accepted 12 December 2008

This paper reports on the development and validation of a real-time reverse transcription-loop-mediated isothermal amplification assay (RT-LAMP) targeting the genomic large RNA segment of Rift Valley fever virus (RVFV). The set of six designed RT-LAMP primers identified strains of RVFV isolated in geographically distinct areas over a period of 50 years; there was no cross-reactivity with other genetically related and unrelated arboviruses. When testing serial sera and plasma from sheep experimentally infected with wild-type RVFV, there was 100% agreement between results of the RT-LAMP, a TaqMan-based real-time PCR, and virus isolation. Similarly, the assay had very high levels of diagnostic sensitivity and specificity when testing various clinical specimens from humans and animals naturally infected with the virus during recent outbreaks of the disease in Africa. The detection of specific viral genome targets in positive clinical specimens was achieved in less than 30 min. As a highly accurate, rapid, and very simple nucleic acid detection format, the RT-LAMP has the potential to be used in less-well-equipped laboratories in Africa and as a portable device during RVF outbreaks in remote areas, and it can be a valuable tool for the differential diagnosis of viral hemorrhagic fevers.

* Corresponding author. Mailing address: Private Bag X4, Sandringham 2131, South Africa. Phone: 27 11 386 6382. Fax: 27 11 882 3741. E-mail: januszp{at}nicd.ac.za

{triangledown} Published ahead of print on 24 December 2008.

Journal of Clinical Microbiology, March 2009, p. 645-651, Vol. 47, No. 3
0095-1137/09/$08.00+0     doi:10.1128/JCM.01412-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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