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Journal of Clinical Microbiology, March 2007, p. 935-941, Vol. 45, No. 3
0095-1137/07/$08.00+0     doi:10.1128/JCM.01258-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Cost-Effective Real-Time Reverse Transcriptase PCR (RT-PCR) To Screen for Dengue Virus followed by Rapid Single-Tube Multiplex RT-PCR for Serotyping of the Virus{triangledown}

Yee-Ling Lai,1 Youne-Kow Chung,2 Hwee-Cheng Tan,1 Hoon-Fang Yap,1 Grace Yap,1 Eng-Eong Ooi,3 and Lee-Ching Ng1*

National Environment Agency, Environmental Health Institute, Singapore,1 National Environment Agency, Environmental Health Department, Singapore,2 DSO National Laboratories, Singapore3

Received 20 June 2006/ Returned for modification 28 August 2006/ Accepted 13 November 2006

Virus detection methodology provides detection of dengue virus in the early phase of the disease. PCR, targeting cDNA derived from viral RNA, has been used as a laboratory-based molecular tool for the detection of Dengue virus. We report the development and use of three real-time one-step reverse transcriptase PCR (RT-PCR) assays to detect dengue cases and serotype the virus involved. The first RT-PCR assay uses SYBR green I as the reporting dye for the purpose of cost-effective screening for dengue virus. The detection limit of the SYBR green I assay was 10 PFU/ml (0.01 equivalent PFU per assay) for all four dengue virus serotypes. The second RT-PCR assay is a duplex fluorogenic probe-based real-time RT-PCR for serotyping clinical samples for dengue viruses. The detection threshold of the probe-based RT-PCR format was 0.1 PFU for serotypes Dengue-1 and Dengue-2, 1 PFU for serotype Dengue-3, and 0.01 PFU for serotype Dengue-4. The third is a fourplex assay that detects any of the four serotypes in a single closed tube with comparable sensitivity. Validation of the assays with local clinical samples collected from 2004 to 2006 revealed that there was an 88% positive correlation between virus isolation and RT-PCR with regard to dengue virus detection and a 100% correlation with seroconversion in subsequent samples. The serotyping results derived from duplex and fourplex assays agree fully with each other and with that derived from immunofluorescence assays.

* Corresponding author. Mailing address: National Environment Agency, Environmental Health Institute, 11 Biopolis Way, 06-05/08 Helios Block, Singapore 138667. Phone: 65 67719108. Fax: 65 67778029. E-mail: ng_lee_ching{at}nea.gov.sg.

{triangledown} Published ahead of print on 10 January 2007.

Journal of Clinical Microbiology, March 2007, p. 935-941, Vol. 45, No. 3
0095-1137/07/$08.00+0     doi:10.1128/JCM.01258-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Das, S., Pingle, M. R., Munoz-Jordan, J., Rundell, M. S., Rondini, S., Granger, K., Chang, G.-J. J., Kelly, E., Spier, E. G., Larone, D., Spitzer, E., Barany, F., Golightly, L. M. (2008). Detection and Serotyping of Dengue Virus in Serum Samples by Multiplex Reverse Transcriptase PCR-Ligase Detection Reaction Assay. J. Clin. Microbiol. 46: 3276-3284 [Abstract] [Full Text]  


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