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J. Clin. Microbiol. doi:10.1128/JCM.00163-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Detection and Serotyping of Dengue Virus in Serum Samples by Multiplex Reverse Transcriptase-PCR/LDR Assay

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; Centers for Disease Control and Prevention, San Juan, Puerto Rico; Centers for Disease Control and Prevention, Fort Collins, CO; Walter Reed Army Institute of Research, Silver Spring, MD; Applied Biosystems, Foster City, CA; Department of Pathology and Laboratory Medicine, Weill Medical College of Cornell University, New York, NY; Department of Pathology, Stony Brook University Medical Center, Stony Brook, NY

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

	Abstract

Detection and successful typing of dengue virus (DENV) from patients with suspected dengue fever is important for the diagnosis of the disease, and the implementation of epidemiologic control measures. A technique for the multiplex detection and typing of DENV serotypes -1 to -4 from clinical samples by PCR/LDR has been developed. A serotype specific PCR reaction amplifies regions of genes C and E simultaneously. The two amplicons are targeted in a multiplex LDR, and the resultant fluorescently labeled ligation products are detected on a universal array. The assay was optimized using 38 DENV strains; and evaluated in 350 archived acute serum samples with a sensitivity of 98.7% and specificity of 98.4%, when compared to real-time PCR. The detection threshold was: 0.017 PFU for DENV-1, 0.004 PFU for DENV-2, 0.8 PFU for DENV-3, and 0.7 PFU for DENV-4. The assay is specific; it does not cross react with other Flaviviruses tested: West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Kunjin virus, Murray Valley virus, Powassan virus, or Yellow fever virus. All but one of 26 genotypic variants of DENV serotypes, of a global dengue virus panel from different geographic regions were successfully identified. The PCR/LDR assay is a rapid, sensitive, specific, and high-throughput technique for the simultaneous detection of all 4 serotypes of DENV.