Development and Evaluation of a SYBR Green-Based Real-Time Multiplex RT-PCR Assay for Simultaneous Detection and Serotyping of Dengue and Chikungunya Viruses.

Abstract

Chikungunya virus (CHIKV) and dengue virus (DENV) have emerged as the two most important arbovirus diseases of global health significance. Similar clinical manifestations, transmission vectors, geographical distribution, and seasonal correlation often result in misdiagnosis of chikungunya infections as dengue cases and vice versa. In this study, we developed a rapid and accurate laboratory confirmative method to simultaneously detect, quantify, and differentiate DENV serotypes 1, 2, 3, and 4 and CHIKV. This SYBR Green I-based one-step multiplex real-time RT-PCR assay is highly sensitive and specific for CHIKV and DENV. Melting temperature analysis of PCR amplicons was used to serotype DENV and to differentiate from CHIKV. The detection limit of the assay was 20, 10, 50, 5, and 10 RNA copies/reaction for DENV-1, DENV-2, DENV-3, DENV-4, and CHIKV, respectively. Our assay did not cross-react with a panel of viruses that included other flaviviruses, alphaviruses, influenza viruses, human enteroviruses, and human coronaviruses. The feasibility of using this assay for clinical diagnosis was evaluated in DENV- and CHIKV-positive patient sera. Accordingly, the assay sensitivity for DENV-1, DENV-2, DENV-3, DENV-4, and CHIKV was 89.66%, 96.67%, 96.67%, 94.12%, and 95.74%, respectively, with 100% specificity. These findings confirmed the potential of our assay to be used as a rapid test for simultaneous detection and serotyping of DENV and CHIKV in clinical samples.

• Authors: Huixin Chen, Mariya Parimelalagan, Yee Ling Lai, Kim Sung Lee, Evelyn Siew-Chuan Koay, Hapuarachchige C Hapuarachchi, Lee Ching Ng, Phui San Ho, Justin Jang Hann Chu
• Journal: The Journal of molecular diagnostics : JMD (J Mol Diagn) Vol. 17 Issue 6 Pg. 722-8 (Nov 2015) ISSN: 1943-7811 [Electronic] United States
• PMID: 26455921 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Chemical References

• Benzothiazoles
• Diamines
• Organic Chemicals
• Quinolines
• RNA, Viral
• SYBR Green I

Topics

• Benzothiazoles
• Chikungunya Fever (virology)
• Chikungunya virus (genetics)
• Dengue (virology)
• Dengue Virus (genetics)
• Diamines
• Humans
• Multiplex Polymerase Chain Reaction (methods)
• Organic Chemicals (chemistry)
• Quinolines
• RNA, Viral (genetics)
• Real-Time Polymerase Chain Reaction (methods)
• Sensitivity and Specificity
• Serotyping