title: 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.affiliation: Laboratory of Molecular RNA Virology and Antiviral Strategies, Department of Microbiology, National University Health System, National University of Singapore, Singapore.
authors.forename: Huixin
authors.initials: H
authors.lastname: Chen
authors.affiliation: School of Applied Science, Republic Polytechnic, Singapore.
authors.forename: Mariya
authors.initials: M
authors.lastname: Parimelalagan
authors.affiliation: Environmental Health Institute, National Environment Agency, Singapore.
authors.forename: Yee Ling
authors.initials: YL
authors.lastname: Lai
authors.affiliation: Environmental Health Institute, National Environment Agency, Singapore.
authors.forename: Kim Sung
authors.initials: KS
authors.lastname: Lee
authors.affiliation: Departments of Laboratory Medicine and Pathology, Molecular Diagnosis Centre, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore.
authors.forename: Evelyn Siew-Chuan
authors.initials: ES
authors.lastname: Koay
authors.affiliation: Environmental Health Institute, National Environment Agency, Singapore.
authors.forename: Hapuarachchige C
authors.initials: HC
authors.lastname: Hapuarachchi
authors.affiliation: Environmental Health Institute, National Environment Agency, Singapore.
authors.forename: Lee Ching
authors.initials: LC
authors.lastname: Ng
authors.affiliation: School of Applied Science, Republic Polytechnic, Singapore. Electronic address: ho_phui_san@rp.edu.sg.
authors.forename: Phui San
authors.initials: PS
authors.lastname: Ho
authors.affiliation: Laboratory of Molecular RNA Virology and Antiviral Strategies, Department of Microbiology, National University Health System, National University of Singapore, Singapore. Electronic address: miccjh@nus.edu.sg.
authors.forename: Justin Jang Hann
authors.initials: JJ
authors.lastname: Chu
mesh_terms: D052160:Benzothiazoles; D065632:Chikungunya Fever / Q000821:virology; D002646:Chikungunya virus / Q000235:genetics*; D003715:Dengue / Q000821:virology; D003716:Dengue Virus / Q000235:genetics*; D003959:Diamines; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009930:Organic Chemicals / Q000737:chemistry*; D011804:Quinolines; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012703:Serotyping
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; C098022:SYBR Green I