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Journal of Clinical Microbiology, November 2007, p. 3506-3513, Vol. 45, No. 11
0095-1137/07/$08.00+0     doi:10.1128/JCM.00936-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Highly Sensitive and Broadly Reactive Quantitative Reverse Transcription-PCR Assay for High-Throughput Detection of Rift Valley Fever Virus{triangledown}

Brian H. Bird,1,2 Darcy A. Bawiec,1 Thomas G. Ksiazek,1 Trevor R. Shoemaker,1 and Stuart T. Nichol1*

Special Pathogens Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, MS G-14, Atlanta, Georgia 30329,1 University of California, Davis, School of Veterinary Medicine, Davis, California 956162

Received 4 May 2007/ Returned for modification 11 July 2007/ Accepted 13 August 2007

Rift Valley fever (RVF) virus is a mosquito-borne virus associated with large-scale epizootics/epidemics throughout Africa and the Arabian peninsula. Virus infection can result in economically disastrous "abortion storms" and high newborn mortality in livestock. Human infections result in a flu-like illness, with 1 to 2% of patients developing severe complications, including encephalitis or hemorrhagic fever with high fatality rates. There is a critical need for a highly sensitive and specific molecular diagnostic assay capable of detecting the natural genetic spectrum of RVF viruses. We report here the establishment of a pan-RVF virus quantitative real-time reverse transcription-PCR assay with high analytical sensitivity (~5 RNA copies of in vitro-transcribed RNA/reaction or ~0.1 PFU of infectious virus/reaction) and efficiency (standard curve slope = –3.66). Based on the alignments of the complete genome sequences of 40 ecologically and biologically diverse virus isolates collected over 56 years (1944 to 2000), the primer and probe annealing sites targeted in this assay are known to be located in highly conserved genomic regions. The performance of this assay relative to serologic assays is illustrated by testing of known RVF case materials obtained during the Saudi Arabia outbreak in 2000. Furthermore, analysis of acute-phase blood samples collected from human patients (25 nonfatal, 8 fatal) during that outbreak revealed that patient viremia at time of presentation at hospital may be a useful prognostic tool in determining patient outcome.

* Corresponding author. Mailing address: Special Pathogens Branch, Division of Viral and Rickettsial Diseases, National Center for Zoonotic, Vector-borne and Enteric Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, MS G-14, Atlanta, GA 30329. Phone: (404) 639-1115. Fax: (404) 639-1118. E-mail: stn1{at}cdc.gov

{triangledown} Published ahead of print on 5 September 2007.

Journal of Clinical Microbiology, November 2007, p. 3506-3513, Vol. 45, No. 11
0095-1137/07/$08.00+0     doi:10.1128/JCM.00936-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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