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Journal of Clinical Microbiology, October 2008, p. 3276-3284, Vol. 46, No. 10
0095-1137/08/$08.00+0 doi:10.1128/JCM.00163-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Detection and Serotyping of Dengue Virus in Serum Samples by Multiplex Reverse Transcriptase PCR-Ligase Detection Reaction Assay
,
S. Das,1
M. R. Pingle,2
J. Muñoz-Jordán,3
M. S. Rundell,2
S. Rondini,1
K. Granger,2
G.-J. J. Chang,4
E. Kelly,5
E. G. Spier,6
D. Larone,7
E. Spitzer,8
F. Barany,2 and
L. M. Golightly1*
Department of Medicine, Division of International Medicine and Infectious Diseases,1 Department of Microbiology and Immunology,2 Department of Pathology and Laboratory Medicine, Weill Medical College of Cornell University, New York,7 Department of Pathology, Stony Brook University Medical Center, Stony Brook, New York,8 Centers for Disease Control and Prevention, San Juan, Puerto Rico,3 Centers for Disease Control and Prevention, Fort Collins, Colorado,4 Walter Reed Army Institute of Research, Silver Spring, Maryland,5 Applied Biosystems, Foster City, California6
Received 25 January 2008/ Returned for modification 14 June 2008/ Accepted 26 July 2008
The detection and successful typing of dengue virus (DENV) from patients with suspected dengue fever is important both for the diagnosis of the disease and for the implementation of epidemiologic control measures. A technique for the multiplex detection and typing of DENV serotypes 1 to 4 (DENV-1 to DENV-4) from clinical samples by PCR-ligase detection reaction (LDR) has been developed. A serotype-specific PCR amplifies the 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 was evaluated with 350 archived acute-phase serum samples. The sensitivity of the assay was 98.7%, and its specificity was 98.4%, relative to the results of 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 the other flaviviruses tested (West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Kunjin virus, Murray Valley virus, Powassan virus, and yellow fever virus). All but 1 of 26 genotypic variants of DENV serotypes in a global DENV 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 four serotypes of DENV.
* Corresponding author. Mailing address: Division of International Medicine and Infectious Diseases, Weill Medical College of Cornell University, 1300 York Avenue, Room A 421, New York, NY 10021. Phone: (212) 746-6320. Fax: (212) 746-8675. E-mail: lgolight{at}med.cornell.edu
Published ahead of print on 6 August 2008.
Supplemental material for this article may be found at http://jcm.asm.org/.
Journal of Clinical Microbiology, October 2008, p. 3276-3284, Vol. 46, No. 10
0095-1137/08/$08.00+0 doi:10.1128/JCM.00163-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
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