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Journal of Clinical Microbiology, November 2001, p. 4119-4124, Vol. 39, No. 11
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.11.4119-4124.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Development and Evaluation of Serotype- and Group-Specific Fluorogenic Reverse Transcriptase PCR (TaqMan) Assays for Dengue Virus

Johnny D. Callahan,1,2,dagger Shuenn-Jue L. Wu,1 Amanda Dion-Schultz,3 Beverly E. Mangold,3 Leonard F. Peruski,3 Douglas M. Watts,4 Kevin R. Porter,5 Gerald R. Murphy,1 Wuryadi Suharyono,6 Chwan-Chuen King,7 Curtis G. Hayes,1 and Joseph J. Temenak1,8,*

Viral and Rickettsial Diseases Department1 and Biological Defense Research Directorate,3 Naval Medical Research Center, and Viral Diseases Department, Walter Reed Army Institute of Research,8 Silver Spring, Maryland 20910-7500; Department of Pathology, University of Maryland, Baltimore, Maryland 212012; Naval Medical Research Center Detachment, American Embassy---Naval Medical Research Center Detachment, APO AA 340314; Naval Medical Research Unit 2, APO AP 96520-81325; National Institute of Health Research and Development, Ministry of Health, Jakarta, Indonesia6; and Institute of Epidemiology, National Taiwan University, Taipei, Taiwan, Republic of China7

Received 13 March 2001/Returned for modification 20 April 2001/Accepted 11 July 2001

Five fluorogenic probe hydrolysis (TaqMan) reverse transcriptase PCR (RT-PCR) assays were developed for serotypes 1 to 4 and group-specific detection of dengue virus. Serotype- and group-specific oligonucleotide primers and fluorogenic probes were designed against conserved regions of the dengue virus genome. The RT-PCR assay is a rapid single-tube method consisting of a 30-min RT step linked to a 45-cycle PCR at 95 and 60°C that generates a fluorogenic signal in positive samples. Assays were initially evaluated against cell culture-derived dengue stock viruses and then with 67 dengue viremic human sera received from Peru, Indonesia, and Taiwan. The TaqMan assays were compared to virus isolation using C6/36 cells followed by an immunofluorescence assay using serotype-specific monoclonal antibodies. Viral titers in sera were determined by plaque assay in Vero cells. The serotype-specific TaqMan RT-PCR assay detected 62 of 67 confirmed dengue virus-positive samples, for a sensitivity of 92.5%, while the group-specific assay detected 66 of 67 confirmed dengue virus-positive samples, for a sensitivity of 98.5%. The TaqMan RT-PCR assays have a specificity of 100% based on the serotype concordance of all assays compared to cell culture isolation and negative results obtained when 21 normal human sera and plasma samples were tested. Our results demonstrate that the dengue virus TaqMan RT-PCR assays may be utilized as rapid, sensitive, and specific screening and serotyping tools for epidemiological studies of dengue virus infections.

* Corresponding author. Present address: Division of Vaccines and Related Products Applications, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, 1401 Rockville Pike, HFM 481, Rockville, MD 20852-1448. Phone: (301) 827-3070. Fax: (301) 827-3532. E-mail: temenak{at}cber.fda.gov.

dagger Present address: Tetracore, Inc., Gaithersburg, MD 20850.

Journal of Clinical Microbiology, November 2001, p. 4119-4124, Vol. 39, No. 11
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.11.4119-4124.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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