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Journal of Clinical Microbiology, June 2003, p. 2408-2416, Vol. 41, No. 6
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.6.2408-2416.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Development of Group- and Serotype-Specific One-Step SYBR Green I-Based Real-Time Reverse Transcription-PCR Assay for Dengue Virus

Pei-Yun Shu, Shu-Fen Chang, Yu-Chung Kuo, Yi-Yun Yueh, Li-Jung Chien, Chien-Lin Sue, Ting-Hsiang Lin, and Jyh-Hsiung Huang*

From Division of Research Development and Laboratory Diagnosis, Center for Disease Control, Department of Health, Taipei, Taiwan, Republic of China

Received 12 November 2002/ Returned for modification 6 January 2003/ Accepted 18 March 2003

A quantitative one-step SYBR Green I-based reverse transcription (RT)-PCR system was developed for the detection and differentiation of four different dengue virus serotypes in acute-phase serum samples. A set of group- and serotype-specific primer pairs was designed against conserved sequences in the core region and evaluated for clinical diagnosis. A linear relationship was obtained between the amount of input RNA and cycle threshold (Ct) value over a range of 10 to 107 PFU per ml of cell culture-derived dengue viruses. The detection limit of the group-specific primer pair was between 4.1 and 43.5 PFU/ml for four dengue serotypes. The detection limit of each of the serotype-specific primer pairs was calculated to be 10 PFU/ml for dengue virus serotype 1 (DEN-1), 4.6 PFU/ml for DEN-2, 4.1 PFU/ml for DEN-3, and 5 PFU/ml for DEN-4. Comparisons between the one-step SYBR Green-based RT-PCR assay and the conventional cell culture method in the clinical diagnosis of dengue virus infection from acute-phase serum samples of confirmed dengue patients were performed. The results showed that 83 and 67% of 193 acute-phase serum samples tested were positive by the one-step SYBR Green-based RT-PCR method and cell culture method, respectively. Further analysis showed that the one-step SYBR Green-based RT-PCR method could detect twice as many acute-phase serum samples with positive dengue-specific immunoglobulin M (IgM) and/or IgG antibodies than cell culture method. Our results demonstrate the potential clinical application of the one-step SYBR Green I-based RT-PCR assay for the detection and differentiation of dengue virus RNA.

* Corresponding author. Mailing address: Division of Research Development and Laboratory Diagnosis, Center for Disease Control, Department of Health, 161, Kun-Yang St., Taipei, Taiwan, Republic of China. Phone: 886-2-26531374. Fax: 886-2-27883992. E-mail: jhhuang{at}cdc.gov.tw.

Journal of Clinical Microbiology, June 2003, p. 2408-2416, Vol. 41, No. 6
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.6.2408-2416.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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