JCM Accepts, published online ahead of print on 21 May 2008

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J. Clin. Microbiol. doi:10.1128/JCM.02335-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Development of a multiplex PCR/LDR assay for detection of West Nile Virus

S. Rondini, M. R. Pingle, S. Das, R. Tesh, M. S. Rundell, J. Hom, S. Stramer, K. Turner, S. N. Rossmann, R. Lanciotti, E. G. Spier, J. Muñoz, D. Larone, E. Spitzer, F. Barany, and L. M. Golightly^*

Department of Medicine, Division of International Medicine and Infectious Diseases, Weill Medical College of Cornell University, New York, NY; Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY; Department of Pathology, University of Texas Medical Branch, Galveston, TX; New York City Dept. of Health and Mental Hygiene, Public Health Laboratory Virology & Immunology Division, New York, NY; Scientific Support Office, American Red Cross Biomedical Services, Gaithersburg, MD; Gulf Coast Regional Blood Center, Houston, TX; Centers for Disease Control and Prevention, Fort Collins, CO; Applied Biosystems, Foster City, CA; Centers for Disease Control and Prevention, San Juan, Puerto Rico; Department of Pathology, Weill Medical College of Cornell University, New York, NY; Department of Pathology, Stony Brook University Medical Center, Stony Brook, N.Y.

^* To whom correspondence should be addressed. Email: Lgolight{at}med.cornell.edu.

We have developed a novel multiplex RT-PCR ligase detection reaction (RT-PCR/LDR) assay for the detection of West Nile virus both in clinical and mosquito pool samples. The method relies on the amplification of three different genomic regions, one in the coding sequence of the non-structural protein NS2a and two in the non-structural protein NS5, to minimize the risk of detection failure due to genetic variation. The sensitivity of the PCR is complemented by the high specificity of the LDR step and the detection of the LDR products can be achieved with capillary electrophoresis (CE), or a Universal DNA microarray. We evaluated the limit of detection for both one-step and two-step multiplex RT-PCR/LDR/CE approaches, which reached, respectively, 0.005 PFU and 0.017 PFU. The assay demonstrated 99% sensitivity when mosquito pool samples were tested, and 100% sensitivity with clinical samples when the one-step approach was employed. The broad strain coverage was confirmed by testing 34 WNV isolates belonging to lineages 1 and 2, and the high specificity of the assay was determined by testing other Flaviviruses as well as negative mosquito pool and clinical samples. In summary, the multiplex RT-PCR/LDR assay could represent a valuable complement to WNV serological diagnosis, especially in early symptomatic patients. In addition, the multiplexing capacity of the technique, which can be coupled to Universal DNA microarray detection, makes it an amenable tool to develop a more comprehensive assay for viral pathogens.

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